Methods of treating hemoglobinopathies

ABSTRACT

Methods of alleviating the symptoms of hemoglobinopathies, including, but not limited to, sickle cell disease, β-thalassemia, and hemoglobin H disease are provided. In some embodiments, the methods comprise administering an inhibitor selected from an ERK inhibitor, a MEK inhibitor, and a Raf inhibitor. Methods of inhibiting adhesion of sickle red blood cells to endothelial cells are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage filing under 35 U.S.C. 371of International Application No. PCT/US2012/35837, filed Apr. 30, 2012,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/480,157, filed Apr. 28, 2011, both of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under K01-DK065040awarded by the National Institutes of Health: National Institute ofDiabetes and Digestive and Kidney Diseases. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .txt format. The .txtfile contains a sequence listing entitled“2012-05-29_5667-00087_Sequence_Listing.txt.” created on May 21, 2012and is 19.8 kilobytes in size. The Sequence Listing contained in this.txt filed is part of the specification and is hereby incorporated byreference herein in its entirety.

BACKGROUND

Vaso-occlusive phenomena and hemolytic anemia are the clinical hallmarksof sickle cell disease (SCD). Sickle (homozygous hemoglobin S, SS) redblood cell (RBC)-based adhesion and vaso-occlusive events likelyinitiate and/or exacerbate the profound vasculopathy present inSCD.^(1, 2) SS RBCs possess unusually active signaling pathways thatcontribute to a panoply of abnormalities, including RBC adhesion to theendothelium and vaso-occlusion.²⁻⁴ Vaso-occlusion results in recurrentpainful episodes and a variety of serious organ system complicationsthat can lead to life-long disabilities and even death.

Cell adhesion is a multistep cellular process that is regulated bycomplex extracellular and intracellular signals, which may differ fromone cell type to another. We have previously shown that abnormal SS RBCinteraction with the endothelium and with leukocytes can be induced viastimulation of β₂ adrenergic receptors (ARs) by the stress hormoneepinephrine.⁴⁻⁵ Such stimulation activates the intracellular cyclicadenosine monophosphate (cAMP)/protein kinase A (PKA) pathway.⁴ βARs areprototypic G protein-coupled receptors (GPCRs), whose signalingproperties are largely mediated by activation of stimulatory GTP-bindingproteins (Gs proteins), which in turn activate adenylate cyclase (AC),leading to generation of cAMP, and the subsequent activation of PKA. ThecAMP/PKA pathway can modulate the mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase (ERKs) cascade.⁷ PKA hasbeen reported to stimulate B-Raf, while inhibiting c-Raf. Therefore, theactivity of downstream signaling proteins, such as MEKs and ERKs, couldbe either enhanced or inhibited depending on the balance of c-Raf andB-Raf activation.^(8, 9) The cellular functions mediated by βARs canalso be independent of adenylyl cyclase activation and involve othermediators instead.^(10, 11)

The functions attributed to ERK1/2 at both cellular and physiologicallevels are diverse, including modulation of proliferation,differentiation, apoptosis, migration, and cell adhesion.¹²⁻¹⁵Physiologically, ERK1/2 is required for immune system development,homeostasis and antigen activation, memory formation, heart development,and responses to many hormones, growth factors and insulin. Most ofthese previous studies have involved only nucleated cells, includingerythroid cells, in which erythropoietin (EPO) is the primary regulatorycytokine of this pathway.¹⁶ However, aberrations in ERK1/2 signaling areknown to occur in a wide range of pathologies, including cancer,diabetes, viral infection, and cardiovascular disease.

SUMMARY

In some embodiments, methods of alleviating at least one symptom of ahemoglobinopathy in a patient is provided. In some embodiments, ahemoglobinopathy is selected from sickle cell disease, β-thalassemia,and hemoglobin H disease. In some embodiments, a hemoglobinopathy issickle cell disease. In some embodiments, at least one symptom isselected from vaso-occlusion, acute painful episodes, chronic hemolysis(aplastic crises), avascular necrosis, infection, end-organ damage, anderythroid hyperplasia.

In some embodiments, methods of inhibiting adhesion of sickle red bloodcells to endothelial cells in a patient are provided. In someembodiments, methods of inhibiting adhesion of sickle red blood cells toleukocytes in a patient are provided. In some embodiments, methods ofinhibiting formation of multicellular aggregates in a patient withsickle cell disease are provided. In some embodiments, methods ofinhibiting adhesion of leukocytes to endothelial cells in a patient withsickle cell disease are provided.

In some embodiments, a method comprises administering at least oneinhibitor selected from a MEK inhibitor, an ERK inhibitor, and a Rafinhibitor.

In some embodiments, the inhibitor is a MEK inhibitor. In someembodiments, the MEK inhibitor is selected from U0126, RDEA119,GSK1120212, PD98059, PD-334581, GDC-0973, CIP-137401, ARRY-162,ARRY-300, PD318088, PD0325901, CI-1040, BMS 777607, AZD8330, AZD6244,AS703026. In some embodiments, the inhibitor is an ERK inhibitor. Insome embodiments, the ERK inhibitor is AEZS-131. In some embodiments,the inhibitor is a Raf inhibitor. In some embodiments, the Raf inhibitoris selected from sorafenib tosylate, GDC-0879, PLX-4720, regorafenib,PLX-4032, SB-590885-R, RAF265, GW5074, XL281, and GSK2118436.

In some embodiments, a method of inhibiting adhesion of sickle red bloodcells to endothelial cells is provided. In some embodiments, a method ofinhibiting adhesion of sickle red blood cells to leukocytes is provided.In some embodiments, a method of inhibiting formation of multicellularaggregates in the presence of sickle red blood cells is provided. Insome embodiments, a method of inhibiting adhesion of leukocytes toendothelial cells in the presence of sickle red blood cells is provided.

In some embodiments, a method comprises contacting sickle red bloodcells with an inhibitor selected from a MEK inhibitor, an ERK inhibitor,and a Raf inhibitor.

In some embodiments, the inhibitor is a MEK inhibitor. In someembodiments, the MEK inhibitor is selected from U0126, RDEA119,GSK1120212, PD98059, PD-334581, GDC-0973, CIP-137401, ARRY-162,ARRY-300, PD318088, PD0325901, CI-1040, BMS 777607, AZD8330, AZD6244,AS703026. In some embodiments, the inhibitor is an ERK inhibitor. Insome embodiments, the ERK inhibitor is AEZS-131. In some embodiments,the inhibitor is a Raf inhibitor. In some embodiments, the Raf inhibitoris selected from sorafenib tosylate, GDC-0879, PLX-4720, regorafenib,PLX-4032, SB-590885-R, RAF265, GW5074, XL281, and GSK2118436.

DESCRIPTION OF THE FIGURES

FIG. 1. ERK undergoes activation in sickle (SS) but not normal red bloodcells (RBCs). A and B. Fifty μg of membrane protein ghosts (SS RBCghosts, n=4, lanes: SS1, SS2, SS3 and SS4; and normal RBC ghosts, n=4,lanes: AA1, AA2, AA3 and AA4) were used per lane. Western blots ofprotein ghosts were stained with antibodies against ERK1/2, glycophorinC as a loading control, and MEK1/2 (n=3 for SS RBC ghosts, lanes: SS1,SS2 and SS3; and n=2 for normal RBC ghosts, lanes: AA1 and AA2). A.ERK1/2 and MEK1/2 are highly expressed in both SS and normal RBCs andare bound to the RBC plasma membrane. B. Quantitative analysis of thedata (normalized according to glycophorin C expression) presented asrelative ERK1/2 expression compared to normal RBCs (p<0.05 for SS vsnormal RBCs, n=4 for each). C and D. Normal RBCs (n=3, lanes: 1, 2, 3and 4) and SS RBCs (n=3, lanes: 5, 6, 7 and 8) were sham-treated (lanes1 and 5), incubated for 1 min with 20 nM epinephrine (epi) (lanes 2 and6), pre-treated with the MEKI, U0126, followed by epi treatment (lanes 4and 8), or treated with U0126 alone (lanes 3 and 7). Mouse 3T3/A31fibroblast lysate was used as a positive control (lane 9). One hundredμg of SS and normal RBC ghost proteins were used per lane. Western blotswere stained with antibodies against ERK and phosphoERK. C. ERK1/2 isphosphorylated at baseline in SS RBCs, and undergoes increasedphosphorylation by epi stimulation. ERK in normal RBCs is notphosphorylated and completely failed to undergo increasedphosphorylation after epi stimulation. D. Quantitative analysis of thedata is presented as fold change in ERK phosphorylation. *p<0.01compared to untreated cells. **p<0.001 compared to epi-treated SS RBCs.E and F. ERK immunoprecipitated from sham-treated (lanes: 1, 2, 5, and6) and epi-treated (lanes: 3, 4, 7, 8, 11, 12, 13 and 14) SS5 RBCs wasincubated without MBP (lanes: 1, 3, 5, 7, 11 and 13) or with MBP (lanes:2, 4, 6, 8, 12 and 14) as a substrate for ERK, with equal proteinamounts per assay condition. Commercial active recombinant human ERK2was incubated without MBP (lanes: 9 and 16) or with MBP (lanes: 10 and15) as negative and positive controls, respectively. E. Immunoblotsindicate that the activity of ERK is conserved and functional in SS RBCsand epi can intensify its activity. SS RBCs obtained from four differentpatients were tested. F. Quantitative analysis of the data is presentedas fold change in ERK phosphorylation (n=4). p=0.0286 compared tonon-treated cells.

FIG. 2. ERK activation in SS RBCs involves the cAMP/PKA pathway and thetyrosine kinase p72^(syk) and is sensitive to the effect of Gα_(i)protein. SS RBCs (panels A, B, C and D), and reticulocyte-enriched and-depleted (mature) SS RBCs (panel E) were sham-treated, treated withforskolin (panel A), epi (panels B and C), the protein kinase Ainhibitor (PKAI), 14-22 amide, (panel B), or Pertussis toxin (PTx)(panels C and D) in the presence or absence of the MEK inhibitor (MEKI)U0126 (panels A, B and D), piceatannol (panel D) or damnacanthal (panelD). RBC proteins were blotted with antibodies against ERK andphosphoERK. Quantitative analysis of the blots is presented as foldchange in ERK phosphorylation. A and B. ERK undergoes phosphorylationvia the cAMP/PKA pathway. A. ERK undergoes increased phosphorylationafter RBC incubation with forskolin, which is inhibited by U0126 (n=3).*p<0.05 compared to untreated cells. **p<0.01 compared toforskolin-treated SS RBCs. B. Phosphorylation of ERK is increased byepi, and this increase was abrogated by either 14-22 amide or U0126(n=3). *p<0.01 compared to untreated cells. **p<0.01 compared toepi-treated SS RBCs. C. ERK phosphorylation in SS RBCs is enhanced byinactivation of the Gα_(i) protein. PTx at either 1 or 2 μg/ml increasedbasal ERK phosphorylation (n=9). *: p<0.001 compared to non-treatedcells; ^(†): p<0.05 compared to epi-treated SS RBCs. D. The tyrosinekinase p72^(syk) is implicated in ERK phosphorylation. PTx at 2 μg/mlupregulated ERK phosphorylation, an effect that was blocked bypiceatannol. Conversely, damnacanthal failed to block ERKphosphorylation induced by PTx (n=3). *p<0.01 compared to untreatedcells. p<0.01 compared to PTx-treated SS RBCs. E. ERK1/2 isphosphorylated at baseline in both reticulocyte-enriched andreticulocyte-depleted (mature) SS RBCs (n=2).

FIG. 3. ERK signaling modulates both SS RBC adhesion to endothelialcells and ICAM-4 phosphorylation. A and B. Activation of ERK signalingup-regulates SS RBC adhesion to the endothelium. SS RBCs weresham-treated, stimulated with epi for 1 min or forskolin, pre-incubatedwith U0126 followed by epi or forskolin, or treated with U0126 alone.Adhesion of SS RBCs to HUVECs was tested in intermittent flow conditionassays. Results are presented as % adherent SS RBCs at a shear stress of2 dynes/cm². Error bars show SEM of four different experiments. A. *:p<0.001 compared to sham-treated; **: p<0.001 compared to epi-treated.B. *: p<0.001 compared to sham-treated; **: p<0.001 compared toforskolin-treated. C and D. The MEK/ERK signaling cascade is involved inICAM-4 (LW) serine phosphorylation. Panel C. Inorganic ³²P radiolabeledintact SS RBCs were incubated in the absence (lane 1) or presence (lanes2, 3, 4, 5 and 6) of serine/threonine protein phosphatase inhibitors(SPI), followed or not (lanes 1 and 2) by treatment with epi (lanes 3,4, 5 and 6). In lanes 4, 5 and 6, SS RBCs were preincubated with PKAI,MEKI or PKAI+MEKI with SPI followed by epi treatment, respectively. Thecpm are representative of three different experiments, calculated bysubtraction of cpm present in a lane (not shown) containingimmunoprecipitates using immunoglobulin P3 from cpm obtained usinganti-LW (ICAM-4) mAb for immunoprecipitation under each set ofconditions indicated. *: p<0.05 and p<0.001 for SPI-treated andSPI+epi-treated vs. sham-treated, respectively; **: p<0.001 compared toSPI+epi-treated SS RBCs. Total LW loaded in each lane was detected usingnitrocellulose membranes of phosphorylated LW blotted with anti-LW mAb.Panel D. SS RBCs were incubated without (lanes 1 and 3) or with epi(lanes 2 and 4). Lanes 1 and 2 were immunoprecipitated with P3. Lanes 3and 4 were immunoprecipitated with anti-LW mAb; all lanes for panel Dwere immunostained with anti-LW mAb.

FIG. 4. SS RBC adhesion is associated with the extent of ERK activation.A and B. Adhesion of SS RBCs to endothelial cells is related to theduration of cell stimulation with epinephrine. Adhesion of RBCs toHUVECs was tested in both intermittent flow and flowing conditionassays, and results are presented as % adherent RBCs at a shear stressof 2 dynes/cm² and number of adherent RBCs/mm², respectively. Normal andSS RBCs were sham-treated, or stimulated with epi for 1 min or 30 min.*: p<0.001 compared to sham-treated SS RBCs; **: p<0.001 compared toepi-treated SS RBCs. Error bars show SEM of four different experiments.C. cAMP production in SS RBCs is associated with the time of cellstimulation with epinephrine. RBCs were treated with IBMX (for basalcAMP levels), followed or not with epi (for 1 min or 30 min) orforskolin. The specific effect of epi and forskolin on cAMP accumulationwas obtained by subtracting basal cAMP levels from the total cAMPlevels. The basal cAMP production and specific amounts of cAMP due toepi or forskolin stimulation were normalized as fmol cAMP/10⁸ RBCs. Dand E. ERK phosphorylation is dependent on the time of SS RBC exposureto epinephrine. SS RBCs were sham-treated or treated with epi for 1 or30 min, U0126, or U0126 followed by epi for 1 or 30 min. Immunoblots ofRBC proteins with antibodies against ERK and phosphoERK (panel D) andquantitative analysis of the data presented as fold change in ERKphosphorylation (panel E) are shown. ERK underwent increasedphosphorylation after 1 min exposure to epi, while phosphorylationdecreased with longer (30 min) cell exposure to epi (n=4). *: p<0.01compared to non-treated cells; **: p<0.01 and p<0.001 for epi-treatedfor 30 min and U0126+epi-treated for 1 min vs cells treated with epi for1 min, respectively (panel E). F. Inorganic ³²P radiolabeled intact SSRBCs were incubated in the presence (lanes 1, 2 and 3) or absence (lane4) of SPI, followed (lanes 2 and 3) or not (lane 1) by treatment withepi for 1 min (lane 2) or 30 min (lane 3). The cpm are representative ofthree different experiments, calculated by subtraction of cpm present ina lane (not shown) containing immunoprecipitates using immunoglobulin P3from cpm obtained using anti-LW (ICAM-4) mAb for immunoprecipitationunder each set of conditions indicated. *: p<0.01 compared tosham-treated; ^(†): p<0.05 compared to SPI+epi (30 min)-treated SS RBCs.G. Prolonged cell exposure to epinephrine negatively affectsphosphorylation of adenylate cyclase-associated protein 1. RBC ghostsisolated from SS and normal RBCs treated with epi for 1 and 30 min wereenriched in phosphopeptides, then subjected to a label-free quantitativephosphoproteomics analysis. Phosphorylation of both serine and threonineon CAP1 in SS RBCs decreased with increased time (1 min vs 30 min) ofcell exposure to epi, while an increase in the abundance of thesephosphopeptides was observed in normal (AA) RBCs after 30 min exposureto epi. Each data point is an average of three analytical replicatemeasurements with error bars indicating standard deviations.

FIG. 5. Phosphorylation of protein 4.1 is induced via the ERK signalingpathway. Sham-treated or U0126-treated SS and normal (AA) RBCs ghostsco-incubated with or without recombinant active ERK2 (ERK2) wereenriched in phosphopeptides, followed by a label-free quantitativephosphoproteomics analysis. Treatment of SS RBCs with U0126 caused asignificant decrease in doubly phosphorylated peptide within protein4.1. Addition of ERK2 to the U0126-treated SS RBC ghosts increased theabundance of this phosphopeptide back to levels observed in untreated SSRBCs. The complementary trend for this phosphorylated peptide was alsoobserved upon the addition of ERK2 to AA RBCs sham-treated orU0126-treated.

FIG. 6. Schematic depiction of proposed increased activation of ERKsignaling pathway in SS RBCs. Epinephrine stimulates β₂ adrenergicreceptors (β₂ARs) on SS RBCs. (β₂ARs are prototypic G_(s)-coupledreceptors (GPCRs), whose signaling is largely mediated by activation ofstimulatory GTP-binding proteins (G_(s) proteins), and inhibited byactivation of Gα_(i) protein. Activation of G_(s) proteins in turnactivates adenylate cyclase (AC), leading to generation of cAMP, and thesubsequent activation of PKA. The activity of downstream signalingproteins, such as MEKs and ERKs is enhanced by PKA activation. Thetyrosine kinase p72^(syk) acts synergistically with PKA to activateMEK/ERK cascade. Activation of ERK results in phosphorylation of the ERKconsensus motif on the cytoskeletal proteins α- and β-adducins, anddematin; and protein 4.1 albeit not at the ERK consensus motif.Phosphorylation of cytoskeletal proteins may result in cytoplasmicmembrane protein conformational changes, which could render ICAM-4accessible to phosphorylation.

FIG. 7. Protein 4.1 levels measured by unbiased label-free quantitativeproteomics normalized to levels in (A) AA RBCs or (B) SS RBCs. AA and SSRBC ghosts, and AA and SS RBC ghosts co-incubated with active ERK2.Protein 4.1 levels remain constant in AA RBCs between the twoconditions, and also in SS RBCs between the two conditions.

FIG. 8. Adhesion of SS RBC to activated-endothelial cells involves ERK.Adhesion of SS RBCs to non-activated and TNF-α activated-HUVECs wastested in intermittent flow condition assays. Results are presented as %cells adherent at a shear stress of 2 dynes/cm² (n=4). Confluentcultures of HUVECs were treated with 10 ng/ml TNF-α for 4 hours, washed,and then tested for their ability to support adhesion of sham-treated SSRBCs or SS RBCs treated with U0126 for 1 hour at 37° C. A. SS RBCs weresham-treated, or treated with 10 μM U0126 MEK inhibitor. U0126significantly inhibited SS RBC adherence to activated endothelial cellsto levels below baseline adhesion of SS RBCs to non-activated HUVECs. *:p<0.001 compared to sham-treated SS RBCs adherent to non-activatedHUVECs; **: p<0.001 compared to sham-treated SS RBCs adherent toTNF-α-activated HUVECs. Error bars show SEM of 4 different experiments.B. SS RBCs were sham-treated, or treated with 1 μM RDEA119, 10 μMRDEA119, 10 μM AZD6244 or 10 μM GSK1120212. All MEK inhibitors blockedSS RBC adhesion to activated HUVECs compared to sham-treated SS RBCadhesion to activated or non-activated HUVECs. *: p<0.0001 compared tosham-treated SS RBC adherent to non-activated or TNF-α-activated HUVECs.Error bars show SEM of 3 different experiments.

FIG. 9. Effect of the MEK inhibitor U0126 on sickle red cell adhesion tothe vascular endothelium and vaso-occlusion in nude mice. Microscopicobservations of postcapillary venules were conducted through implanteddorsal skin-fold window chambers after infusion of human sickle RBCsinto the tail vein of nude mice using 20× magnification. Vessels withoutadherent cells appear gray, due to the fluorescence of rapidly movingRBCs. TNF-α was injected prior to infusion of the MEK inhibitor U0126 orhuman sickle RBCs, to induce inflammation, since sickle red celladhesion to activated endothelium in vitro is markedly up-regulatedcompared to sickle cell adhesion to non-activated endothelium. A. Mice(n=5) were injected with 0.02 mg/kg TNF-α intraperitoneally 4 hoursbefore infusion into the tail vein of fluorescently-labeled human sickleRBCs. Human sickle RBCs showed striking adhesion to postcapillaryvenules, with permanent vaso-occlusion at junctions, although it wasalso observed in straight non-junctional venular segments as indicatedby arrows. B. Mice (n=5) were injected with 0.02 mg/kg TNF-αintraperitoneally 3 hours and 30 min before infusion into the tail veinof placebo (vehicle, 0.4% dimethyl sulfoxide (DMSO) in normal saline).Thirty minutes later, placebo-treated mice were infused into the tailvein with fluorescently-labeled human sickle RBCs. Infusion of humansickle RBCs resulted in marked RBC adhesion to postcapillaryendothelium, with intermittent occlusion of vessels and permanentblockage of some vessel segments, especially at junctions. These dataindicate that intravenous administration of placebo did not preventhuman sickle red blood cell adhesion and vasoocclusion. C and D. Micewere injected with 0.02 mg/kg TNF-α intraperitoneally 3 hours and 30 minbefore infusion into the tail vein of U0126 (2 mg/kg in 0.4% DMSO innormal saline, n=5) (C), or U0126 (0.2 mg/kg in 0.04% DMSO in normalsaline, n=1) (D). Thirty minutes later, U0126-treated mice were infusedwith fluorescently-labeled human sickle RBCs. Human sickle RBCs showedlittle adhesion, which was observed only in few venular segments withoutpromotion of frank vaso-occlusion as indicated by arrows. Scale bar=150μm. E. Effect of U0126 on % venular length occupied by SS RBCs. Videoframes showing >30 vessel segments were used to quantify the length ofvenules occupied by SS RBCs in animals treated as described in Panels B,C and D. The values were averaged among groups of animals to representthe mean % venular length occupied by SS RBCs. Error bars show SEM. *:p<0.001 vs placebo for vessel diameter ≦25 μm; **: p<0.05 vs placebo forvessel diameter >25 μm.

FIG. 10. MEK inhibitors prevent SSRBC adhesion in inflammed vessels andvaso-occlusion in vivo. Human sickle RBCs were sham-treated or treatedwith the MEK inhibitor RDEA119 or U0126 ex vivo, washed extensively, andthen infused into the tail vein of nude mice pretreated with TNF-α.Microscopic observations of venules were conducted through a dorsalskin-fold window chamber using 10× and 20× magnifications, afterinfusion of fluorescently labeled human SS RBCs. Vessels withoutadherent cells appear gray, due to rapidly moving fluorescent RBCs. FIG.10A. Infusion of sham-treated SS RBCs (n=5) resulted in marked RBCadherence in inflammed venules and vaso-occlusion as shown by arrows.FIG. 10B. U0126-treated SS RBCs (n=5) show rare adhesion in inflammedvessels as indicated by arrows, but no apparent vaso-occlusion. FIG.10C. Treatment of human SSRBCs with RDEA119 (n=5) dramatically decreasedhuman SS RBC adhesion as indicated by arrows and prevented vesselobstruction. Scale bar=150 μm. FIG. 10D. Effect of RDEA119 treatment ofSS RBCs on % venular length occupied by treated SS RBCs. Video framesshowing >30 vessel segments were used to quantify the length of venulesoccupied by SS RBCs in animals treated as described above. The valueswere averaged among groups of animals to represent the mean % venularlength occupied by SS RBCs. Error bars show SEM of 5 differentexperiments for each treatment. *: p<0.001 vs sham for vessel diameter≦25 μm; **: p<0.05 vs sham for vessel diameter >25 μm.

FIG. 11. MEK inhibitors inhibit epinephrine-stimulated 55 RBC-mediatingneutrophil adhesion to endothelial cells. SS RBCs were treated withepinephrine (Epi), or pre-incubated with U0126, RDEA119, AZD6244 orGSK1120212 followed by treatment with Epi, and then washed. Nativeneutrophils (PMNs) from healthy donors were then co-incubated withtreated-SS RBCs, and assayed for their ability to adhere to HUVECs.Adhesion of PMNs was much higher when cells were co-incubated withEpi-treated SS RBCs compared to adhesion of naïve PMNs not co-incubatedwith SS RBCs. However, all MEK inhibitors tested were equally able tomarkedly reduce the effect of epinephrine-stimulated SS RBCs on PMNadhesion to HUVECs when compared to adhesion of epinephrine-activated SSRBC-mediated PMN adhesion. *: p<0.0001 compared to native PMNs; and **:p<0.0001 compared to PMN adhesion mediated by epinephrine-treated SSRBCs. Error bars show SEM of 4 different experiments.

FIG. 12. (A) AScore site localization scoring distributions across all375 unique phosphorylated peptides from RBC ghost preparations. (B)Coefficient of variation (% CV) distribution of measured phosphopeptidepeak heights from triplicate LC-MS analysis of a treatment groupfollowing accurate-mass and retention time alignment. Error barsindicate standard deviation within each % CV bin across all eighttreatment groups.

FIG. 13. Selected ion chromatogram (A) and AUC quantitation (B) of173-VADPDHDHTGFLTE[pY]VATR-191 ([M+3H]³⁺ 741.999 m/z) (SEQ ID NO:1) theactive form of Mitogen-Activated Protein Kinase-1 (GN=MAPK1, ERK2),across 24 LC-MS injections. This peptide was qualitatively identifiedwith a maximum mascot ion score of 63.3 and a site localization Ascoreof 41+/−12.

FIG. 14. A set of graphs is showing the distribution of types ofphosphorylated residues and phosphorylated residues per peptide (A) andthe biological functions of the phosphorylated proteins (B).

FIG. 15. Two-dimensional (2D) agglomerative cluster analysis of Z-scoretransformed (ie magnitude of significance of change) phosphopeptideintensities across eight unique RBC treatment groups. Personcorrelations were used as the measure of similarity (−1 dissimilar, +1identical). (A) RBC membrane fractions from healthy (AA) and sickle-cell(SS) patients pre-treated with or without the MEK inhibitor, U0126, andsubsequently with or without activated ERK2. (B) Analysis performed onlyon SS or AA RBC treatment groups.

FIG. 16. ERK1/2 signaling modulates glycophorin A serinephosphorylation. Inorganic ³²P radiolabeled intact SS RBCs wereincubated in the absence (lane 1) or presence (lanes 2, 3 and 4) ofserine/threonine protein phosphatase inhibitors (SPI), not followed(lanes 1 and 2) or followed by treatment with epinephrine (epi) (lanes 3and 4). In lane 4, SS RBCs were preincubated with MEK1/2 inhibitor U0126prior to treatment with SPI followed by epi treatment. The cpm arerepresentative of three different experiments, calculated by subtractionof cpm present in a lane (not shown) containing immunoprecipitates usingimmunoglobulin P3 from cpm obtained using anti-glycophorin A mAb forimmunoprecipitation under each set of conditions indicated. *: p<0.05and p<0.001 for SPI-treated and SPI+epi-treated vs. sham-treated,respectively; **: p<0.001 compared to SPI+epi-treated SS RBCs. Totalglycophorin A loaded in each lane was detected using nitrocellulosemembranes of phosphorylated glycophorin A blotted with anti-glycophorinA mAb. PhosphorImager analysis of immunoprecipitated ³²P-radiolabeledglycophorin A and negative control immune complexes showed thatglycophorin A of non-stimulated SS RBCs (FIG. 2, lane 1) is modestlyphosphorylated at baseline. Treatment of SS RBCs with serine phosphataseinhibitors (SPI) (lane 2) slightly increased glycophorin Aphosphorylation by 1.9±0.1-fold (p<0.05, n=3), suggesting that increasedglycophorin A phosphorylation is a result of serine phosphorylation, astyrosine phosphatase inhibitors were not present. Epinephrine in thepresence of SPI had a stronger effect on glycophorin A phosphorylation(2.93±0.35-fold increase over sham-treated SS RBCs; p<0.001) (lane 3).However, treatment of SS RBCs with the MEK/12 inhibitor U0126significantly decreased the combined effect of epinephrine and SPI onglycophorin A phosphorylation (lane 4) compared to cells treated withepinephrine and SPI (p<0.001) (lane 3).

DETAILED DESCRIPTION

Preliminary studies have suggested that the mitogen-activated proteinkinase (MAPK)/the extracellular signal-regulated kinase (ERK1/2) ispresent at higher abundance in sickle red blood cells (SS RBCs) than innormal RBCs and is bound to the cytoplasmic membrane. The presentinventors have shown that RK1/2 is active in enucleated SS RBCs, andthat triggering this kinase promotes activation of signaling pathwaysand consequent RBC adhesion to the endothelium. Stimulation of β₂adrenergic receptors (β₂ARs) on SS RBCs by epinephrine for a briefperiod of time increases activation of the ERK1/2 signaling cascade,which is involved in phosphorylation of the RBC adhesion receptor ICAM-4and protein 4.1. The present inventors also found that the ERK consensusmotifs on dematin and α- and β-adducins undergo increased serinephosphorylation, indicating that these cytoskeletal proteins aresubstrates for ERK.

ERK has been implicated in erythropoietin-induced erythroid cellproliferation and survival,²⁹ and the present inventors have nowdemonstrated that the activity of this kinase and its upstream signalare conserved in mature SS RBCs. In some instances, ERK1/2 ishyperactive without stimulation of SS RBCs, and increased activation ofthis kinase can increase within 1 minute of SS RBC exposure toepinephrine. In contrast, in normal RBCs, despite the abundance ofERK1/2, ERK is not active at baseline and fails to becomephosphorylated/activated with epinephrine or forskolin stimulation. Theinability of ERK1/2 to undergo activation in normal RBCs suggests thatthe activity of ERK itself and/or at least one of the upstream effectorsrequired for ERK activation is lost. Indeed, investigators havepreviously described that RBCs undergo maturation-related loss ofmultiple protein kinase activities, including PKA, PKC, and caseinkinases.³⁰ In contrast, although SS RBCs are also fully differentiated,the present inventors have found that preservation of ERK activity andits downstream signaling molecules appears to be involved at least inthe abnormal activation of RBC adhesive function.

Our data further implicate involvement of the protein G_(s) and cAMP/PKAas upstream mediators in activation of ERK and its downstream signaltransduction pathway. Our findings are consistent with studies bySchmitt and Stork⁷ demonstrating that isoproterenol stimulation ofendogenous β₂ARs activated ERK in HEK293 cells via a cAMP-dependent PKApathway, and this ERK pathway was insensitive to the effect of PTx,which inactivates the protein Gα_(i). In addition to PKA, we have alsoidentified a role for the tyrosine kinase p72^(Syk) in activation of ERKin SS RBCs, while excluding involvement of p56^(Syk)-related Src familytyrosine kinases. Thus, in SS RBCs, PKA and the tyrosine kinasep72^(Syk) are implicated in ERK activation, acting most likely inconcert to regulate the MEK/ERK signaling pathway.

The engagement of epinephrine-stimulated ERK in regulation of SS RBCadhesion to the endothelium suggests that the MEK/ERK signal can promotean adhesive, vaso-occlusive pathology. It is also apparent from the dataherein that epinephrine-induced adhesion of SS RBCs to non-activatedendothelial cells requires ICAM-4 phosphorylation, which occurs via thecAMP/PKA/ERK signaling pathway. Furthermore, the adhesive function of SSRBCs appeared to be related to the extent of ERK and ICAM-4phosphorylation/activation, since all three similarly increased ordecreased depending on the time of cell exposure to epinephrine.Additionally, basal cAMP levels, the upstream effector of MEK/ERK, weremuch higher in SS RBCs than in normal cells, suggesting that theincreased level of cAMP in SS RBCs reflects at least in part thepersistence of the abnormal ERK activation and RBC adhesive phenotype.However, although epinephrine increased cAMP levels in only 50% of theSCD patient samples tested, cAMP production, which seems to be needed toactivate ERK signaling in these sickle cells, was also influenced by theduration of cell exposure to epinephrine. This may be explained at leastin part by the dramatic decrease in the abundance of phosphopeptideswithin CAP1 in SS RBCs due to continued cell exposure to epinephrinestimulation. PKA might also exert a negative feedback loop throughactivation of phosphodiesterases, resulting in cAMP hydrolysis switchingoff downstream signaling because of the extended cell exposure toepinephrine (Rochais F, J Biol Chem. 2004). CAPs are not only involvedin adenylate cyclase (AC) association, but in actin binding, SH3binding, and cell morphology maintenance as well (Hubberstey A V, FASEB,2002; and Bertling E, Mol. Biol. Cell, 2004). Previous observations ofincreased normal RBC membrane filterability after epinephrine treatmentfor 20 min (Tuvia S, J. Physiol., 1999), explain the enhancedphosphorylated CAP1 in normal RBCs after 30 min epinephrine exposure.Furthermore, Shain et al.³¹ suggested that maintenance of altered cellmorphology required persistent increased cAMP levels due to continuousPAR stimulation. In contrast, our data suggest that when an increase inERK activation occurs within 1 min of cell exposure to epinephrine,persistent β₂AR stimulation has a negative effect on ERK activation andconsequently the RBC adhesive function. Based on this analysis, it isexpected that inhibition of b-Raf or c-Raf will result in similareffects in SS RBCs as these are additional upstream activators in thispathway.

The data herein also define the putative downstream targets of ERK inRBCs. Label-free quantitative phosphoproteomics analysis implicates ERK2in phosphorylation of protein 4.1 and shows that the ERK consensusmotifs on dematin and adducins α and β undergo increased phosphorylationin the presence of this kinase. Dematin is also a substrate for PKC andPKA, and PKA-induced dematin phosphorylation completely abolishes itsactin bundling capability.^(32, 33) Alternatively, rapid phosphorylationof α- and β-adducins by PKC at Ser-726 and Ser-713, respectively (MannoS, J Biol Chem, 2005) leads to decreased F-actin capping anddissociation of spectrin from actin, implicating adducin phosphorylationin cytoskeletal remodeling.³⁴ Furthermore, studies have previously shownthat protein 4.1 phosphorylation, induced by cAMP-dependent kinase atSer-331 and protein kinase C at Ser-312 documented after 20 min of cellstimulation (Manno S, J Biol Chem, 2005), results in a significantreduction in both the ability of protein 4.1 to promote spectrin bindingto F-actin and in spectrin-protein 4.1 binding.³⁵ Thus, phosphorylationof cytoskeletal proteins and proteins of the junctional complexes by ERKin SS RBCs may also lead to cytoskeletal deorganization, which in turn,could potentially render ICAM-4 accessible to undergo phosphorylation,and to then mediate adhesion to the endothelium, or to affect itsadhesivity with an as yet undetermined kinase. In fact, otherinvestigators have shown that cell adhesion can be regulated by anintricate network of signaling molecules, which are responsible forguiding their interaction with substrate mainly via cytoskeletonrearrangement.³⁶ A schematic overview of the proposed β₂-AR signalingpathway in SS RBCs is shown in FIG. 6.

Finally, while aberrant ERK activation may arise in other pathologies,the present inventors are the first to describe atypical ERK activationin SS RBCs and its involvement in the abnormal RBC adhesion to theendothelium. Abnormal activation of ERK in SS RBCs may therefore beassociated with the pathophysiology of sickle cell disease, making theMEK/ERK pathway a therapeutic target for preventing and treatingvaso-occlusion. Various MEK and ERK inhibitors are currently beinginvestigated in phase II clinical trials as therapeutic agents incancer. The present invention provides methods of alleviating thesymptoms of hemoglobinopathies, such as sickle cell disease andβ-thalassemia, comprising administering MEK and/or ERK inhibitors.

The phospho-proteomic analysis presented in the Examples suggests thataberrant ERK activation may also be involved in additional symptoms andRBC defects associated with sickle cell disease. SS RBCs arecharacterized by a panoply of abnormalities, including polymerization ofdeoxygenated HbS, persistent oxidative membrane damage associated withHbS cyclic polymerization, abnormal activation of membrane cationtransports, cell dehydration, and cytoskeletal dysfunction. Inparticular, the Examples demonstrate that ERK alters the phosphorylationstate of proteins that may be involved in maintaining mechanicalstability of RBC and may lead to a reduction in shear resistance as wellas effect RBC shape, flexibility, anion transport and proteintrafficking. Thus, MEK/ERK inhibition may result not only inamelioration of vaso-occlusion, but also other symptoms of sickle celldisease.

DEFINITIONS

The subject matter disclosed herein is described using severaldefinitions, as set forth below and throughout the application.

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below, itis to be understood that as used in the specification, embodiments, andin the claims, “a”, “an”, and “the” can mean one or more, depending uponthe context in which it is used.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” or“approximately” will mean up to plus or minus 10% of the particular termand “substantially” and “significantly” will mean more than plus orminus 10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.”

As used herein, the terms “patient” and “subject” may be usedinterchangeably and refer to one who receives medical care, attention ortreatment. As used herein, the term is meant to encompass a persondiagnosed with a disease such as a hemoglobinopathy or at risk fordeveloping a hemoglobinopathy (e.g., a person who may be geneticallyhomozygous or heterozygous for a sickle cell-causing mutation, but isnot symptomatic). A “patient in need thereof” may include a patienthaving, suspected of having, or at risk for developing ahemoglobinopathy or symptoms thereof.

As used herein, the term “treatment,” “treating,” or “treat” refers tocare by procedures or application that are intended to alleviatesymptoms of a disease (including reducing the occurrence of symptoms ofthe disease). Although it is preferred that treating a condition ordisease such as a hemoglobinopathy will result in an improvement of thecondition, the term treating as used herein does not indicate, imply, orrequire that the procedures or applications are at all successful inalleviating symptoms associated with any particular condition. Treatinga patient may result in adverse side effects or even a worsening of thecondition which the treatment was intended to improve. Treating mayinclude treating a patient having, suspected of having, or at risk fordeveloping a hemoglobinopathy or symptoms thereof.

As used herein the term “effective amount” refers to the amount or doseof the agent, upon single or multiple dose administration to thesubject, which provides the desired effect in the subject underdiagnosis or treatment. The disclosed methods may include administeringan effective amount of the disclosed agents (e.g., as present in apharmaceutical composition) for treating a hemoglobinopathy in thepatient, whereby the effective amount alleviates symptoms of thehemoglobinopathy (including reducing the occurrence of symptoms of thehemoglobinopathy).

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of agent administered, a numberof factors can be considered by the attending diagnostician, such as:the species of the patient; its size, age, and general health; theparticular symptoms or the severity of the hemoglobinopathy; theresponse of the individual patient; the particular agent administered;the mode of administration; the bioavailability characteristics of thepreparation administered; the dose regimen selected; the use ofconcomitant medication; and other relevant circumstances.

The phrase “alleviates at least one symptom,” as used herein, means thata particular treatment results in a lessening of at least one symptom ofa disease. Such lessening of a symptom may be a qualitative orquantitative reduction in the severity of the symptom, or may be areduction in the number of occurrences of the symptom; even though eachoccurrence may be as severe as it was before the treatment (one or moreoccurrences may also be less severe). Nonlimiting exemplary symptoms ofsickle cells disease include vaso-occlusion, acute painful episodes,chronic hemolysis (aplastic crises), avascular necrosis, infection,end-organ damage, acute chest syndrome, leg ulceration, priapism, anddecreased life expectancy. Nonlimiting exemplary symptoms of thalassemiainclude hemolysis, erythroid hyperplasia, biliary tract disease,infection, leg ulcers, extramedullary hematopoiesis, increased risk fordeveloping thromboembolic phenomena, liver and heart damage, anddecreased life expectancy.

The term “hemoglobinopathy,” as used herein, refers to a condition thatis caused by a genetic mutation in a globin gene that results in amutated hemoglobin α chain or β chain protein, or a condition that iscaused by a genetic mutation that results in an abnormal ratio ofhemoglobin α chain to β chain or crossover fusion products of 2 globingenes. Nonlimiting exemplary hemoglobinopathies include sickle celldisease (including, but not limited to, homozygous for hemoglobin S anda variety of sickle cell syndromes that result from inheritance of thesickle cell gene in compound heterozygosity with other mutant betaglobin genes, including, but not limited to, hemoglobin SC disease(HbSC), sickle beta(+) thalassemia, sickle beta(0) thalassemia, sicklealpha thalassemia, sickle delta beta(0) thalassemia, sickle Hb Lepore,sickle HbD, sickle HbO Arab, and sickle HbE), β-thalassemia (including,but not limited to, β-thalassemia major (also known as Cooley's anemia)and β-thalassemia intermedia, and hemoglobin H disease (α-thalassemiawith α⁺-α⁰ phenotype)). Nonlimiting exemplary genetic mutations thatcause sickle cell disease include Hb SS, which is hemoglobin with an E6Vmutation in each of the two hemoglobin β chains; Hb SC, which ishemoglobin with one β chain with an E6V mutation and one β chain with anE6K mutation; Hb SD, which is hemoglobin with one β chain with an E6Vmutation and one β chain with a β121 Glu→Gln mutation; sickle-HbO Arab,which is hemoglobin with one β chain with an E6V mutation and one βchain with a β121(GH4)gGlu→Lys mutation; and Hb SE, which is hemoglobinwith one β chain with an E6V mutation and one β chain with an E26Kmutation. Nonlimiting exemplary genetic mutations that causeβ-thalassemia include various β-mutations, such as IVS II-I, CD 36/37,CD41/42, CD 39; IVS1-6; IVS1-110, CD71/72, IVS1-5, IVS1-1, CD26,IVS2-654, CAP+1, CD19, -28, -29, IVS1-2, InCD (T-G) and CD17; and rareβ-mutations, i.e. InCD (A-C), CD8/9, CD43, -86, CD15, Poly A. Poly T/C,IVS2-1, CD1, CD35/36, CD27/28, CD16, CD37, and 619bpDEL. Nonlimitingexemplary genetic mutations that cause Hb H disease include α⁺-α⁰phenotypes such as α2 Poly A (AATAAA→AATA-), α2 Poly A (AATAAA→AATGAA),and α2 Poly A (AATAAA→AATAAG); α⁺ phenotypes such as α2 CD 142(TAA→CAA), α2 CD 142 (TAA→AAA), and α2 CD 142 (TAA→TAT); and α⁰phenotypes such as—α³² Init CD (ATG→GTG), -^(SEA), -^(YHAI), -^(MED II),-^(BRIY), -^(MED I), -^(SA), -(α)^(20.5), and -^(FIL).

The term “MEK inhibitor,” as used herein, refers to an inhibitor of MEKkinase activity. A MEK inhibitor may be any type of molecule, includingbut not limited to, small molecules and expression modulators (such asantisense molecules, microRNAs, siRNAs, etc.), and may act directly onthe MEK protein, may interfere with expression of the MEK protein (e.g.,transcription, splicing, translation, and/or post-translationalprocessing), and/or may prevent proper intracellular localization of theMEK protein. Exemplary MEK inhibitors include, but are not limited to,U0126, PD98059, PD-334581, GDC-0973, CIP-137401, ARRY-162, ARRY-300,PD318088, PD0325901, CI-1040, BMS 777607, AZD8330, AZD6244, RDEA119,GSK1120212 and AS703026.

The term “ERK inhibitor,” as used herein, refers to an inhibitor of ERKkinase activity. An ERK inhibitor may be any type of molecule,including, but not limited to, small molecules and expression modulators(such as antisense molecules, microRNAs, siRNAs, etc.), and may actdirectly on the ERK protein, may interfere with expression of the ERKprotein (e.g., transcription, splicing, translation, and/orpost-translational processing), and/or may prevent proper intracellularlocalization of the ERK protein. A nonlimiting exemplary ERK inhibitoris AEZS-131.

The term “Raf inhibitor,” as used herein, refers to an inhibitor ofb-Raf kinase activity and/or c-Raf kinase activity. A Raf inhibitor maybe any type of molecule, including, but not limited to, small moleculesand expression modulators (such as antisense molecules, microRNAs,siRNAs, etc.), and may act directly on the Raf protein, may interferewith expression of the Raf protein (e.g., transcription, splicing,translation, and/or post-translational processing), and/or may preventproper intracellular localization of the Raf protein. Nonlimitingexemplary Raf inhibitors include sorafenib tosylate, GDC-0879, PLX-4720,regorafenib, PLX-4032, SB-590885-R, RAF265, GW5074, XL281, andGSK2118436.

A table providing additional information on some of the exemplified MEK,ERK, and B-Raf inhibitors is provided below as Table 4.

TABLE 4 Non-limiting exemplary inhibitors of MEK, ERK, and/or B-RafInhibitor Alternate name(s) Structure or source U0126 U0126-EtOH

PD98059

PD-334581

GDC-0973 XL518 Genentech CIP-137401 CIP-1374 Allostem TherapeuticsARRY-162 Array BioPharma/Novartis ARRY-300 Array BioPharma/NovartisPD318088

PD0325901

CI-1040 PD184352

BMS 777607

AZD8330 ARRY-424704 ARRY-704

AZD6244 Selumetinib

AS703026 MSC1936369B

AEZS-131 Aeterna Zentaris Inc. sorafenib tosylate BAY 43-9006 AZ 628

GDC-0879

PLX-4720

regorafenib BAY 73-4506

PLX-4032 RG7204

SB-590885

RAF265 CHIR-265

GW5074

XL281 BMS-908662 Exelixis GSK2118436 GlaxoSmithKline

In some embodiments, methods of alleviating at least one symptom of ahemoglobinopathy in a patient are provided. Such methods comprise, insome embodiments, administering to the patient an inhibitor selectedfrom a MEK inhibitor, an ERK inhibitor, and a Raf inhibitor. Nonlimitingexemplary hemoglobinopathies include β-thalassemia, sickle cell diseaseand Hemoglobin H.

For the treatment of sickle cell disease or other hemoglobinopathies, insome embodiments, at least one symptom that may be alleviated byadministering the inhibitors described herein is selected fromvaso-occlusion, acute painful episodes, chronic hemolysis (aplasticcrises), avascular necrosis, infection, end-organ damage, and erythroidhyperplasia. In some embodiments, alleviating a symptom of sickle celldisease means reducing the amount, frequency, duration or severity ofthe symptom. For example, for vaso-occlusion, in some embodiments,alleviating the symptom includes reducing the average size of thevaso-occlusions and/or reducing the number of vaso-occlusions. Further,alleviating a symptom may or may not result in a reduction in thediscomfort experienced by the patient as a result of the symptom. Thatis, in some embodiments, while the number and/or average size ofvaso-occlusions may be reduced following a treatment described herein,the patient may or may not experience a similar reduction in acute paincaused by vaso-occlusion.

In some embodiments, when vaso-occlusion is alleviated by administrationof an inhibitor described herein, acute painful episodes are alsoalleviated (i.e., the number and/or severity is reduced). In someembodiments, when vaso-occlusion is alleviated by administration of aninhibitor described herein, hemolysis is also alleviated. In someembodiments, vascular endothelial injury is alleviated by administrationof an inhibitor described herein. In some embodiments, when hemolysis isalleviated by administration of an inhibitor described herein, theincidence of infections is reduced. In some embodiments, when hemolysisis alleviated by administration of an inhibitor described herein,erythroid hyperplasia is also alleviated. In some embodiments, whenvaso-occlusion and/or hemolysis are alleviated by administration of aninhibitor described herein, end-organ damage is also alleviated.

In some embodiments, methods of inhibiting adhesion of sickle red bloodcells to endothelial cells are provided. In some embodiments, methods ofinhibiting adhesion of sickle red blood cells to leukocytes areprovided. Such methods comprise, in some embodiments, contacting thesickle red blood cells with an inhibitor selected from a MEK inhibitor,an ERK inhibitor, and a Raf inhibitor.

In some embodiments, methods of inhibiting adhesion of sickle red bloodcells to endothelial cells in a patient are provided. In someembodiments, methods of inhibiting adhesion of sickle red blood cells toleukocytes in a patient are provided. Such methods comprise, in someembodiments, administering to the patient an inhibitor selected from aMEK inhibitor, an ERK inhibitor, and a Raf inhibitor.

In some embodiments, a method comprises administering to the patient, orcontacting a sickle red blood cell with, a MEK inhibitor. Nonlimitingexemplary MEK inhibitors include U0126, PD98059, PD-334581, GDC-0973,CIP-137401, ARRY-162, ARRY-300, PD318088, PD0325901, CI-1040, BMS777607, AZD8330, AZD6244, RDEA119, GSK1120212 and AS703026. In someembodiments, a method comprises administering to the patient, orcontacting a sickle red blood cell with, an ERK inhibitor. A nonlimitingexemplary ERK inhibitor is AEZS-131. In some embodiments, a methodcomprises administering to the patient, or contacting a sickle red bloodcell with, a Raf inhibitor. In some embodiments, the Raf Inhibitorinhibits b-RAF. In some embodiments, the Raf inhibitor inhibits c-Raf.In some embodiments, the Raf inhibitor inhibits both b-Raf and c-Raf.Nonlimiting exemplary Raf inhibitors include sorafenib tosylate,GDC-0879, PLX-4720, regorafenib, PLX-4032, SB-590885-R, RAF265, GW5074,XL281, and GSK2118436.

In some embodiments, a method comprises administering to the patient, orcontacting a sickle red blood cell with a combination of two or moreinhibitors selected from a MEK inhibitor, an ERK inhibitor, and a Rafinhibitor. The two or more inhibitors may be co-administered.Co-administration indicates the inhibitors may be administered in anyorder, at the same time or as part of a unitary composition. The twoinhibitors may be administered such that one inhibitor is administeredbefore the other with a difference in administration time of 1 hour, 2hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4days, 7 days, 2 weeks, 4 weeks or more.

Administration to a subject may include formulating the therapeuticagents, such as a MEK inhibitor, an ERK inhibitor, and/or a B-Rafinhibitor, with pharmaceutically acceptable carriers and/or excipients,etc., to form pharmaceutical compositions. Suitable formulations fortherapeutic compounds are available to those skilled in the art.Administration may be carried out by any suitable method, includingintraperitoneal, intravenous, intramuscular, intrathecal, subcutaneous,transcutaneous, oral, nasopharyngeal, or transmucosal absorption amongothers. The dosage for a particular subject may be determined based on,for example, the subject's weight, height, and/or age; the severity ofthe subject's disease or symptoms; the length of treatment and/or numberof doses anticipated in a particular regiment; the route ofadministration; etc.

The following examples are illustrative and are not intended to limitthe disclosed subject matter. All references cited herein areincorporated herein by reference in their entireties.

EXAMPLES Example 1 Materials and Methods

Endothelial Cells.

Primary human umbilical vein endothelial cells (HUVECs) were grown asmonolayers in EBM2 medium (Lonza Walkersville, Inc., Walkersville, Md.)supplemented with EGM2 (Lonza Walkersville) as described previously.⁴ ECpassage was accomplished with trypsinization, as required. Cells wereused until they reached the 5th passage. For flow chamber experiments,HUVECs were cultured until they reached confluence on clear glass slidesprecoated with 2% gelatin.

Antibodies.

Antibodies used included the following monoclonal and polyclonalantibodies (Abs, as purified immunoglobulin [Ig] unless otherwisenoted): BS46 (mouse anti-ICAM-4, generously provided by Dr. Jean-PierreCartron, INSERM Unité 665, Paris, France);¹⁷ and mouseanti-phospho-myelin basic protein (Millipore, Temecula, Calif.); mouseanti-human transferrin receptor (BD Biosciences, San Jose, Calif.); andmouse anti-human glycophorin C produced in our laboratory. Rabbitanti-human ERK1/2 was from Upstate, Charlottesville, Va.; rabbitanti-human phospho-ERK1/2 was from Cell Signaling Technology, Danvers,Mass.; and rabbit anti-human MAPK kinase (MEK1/2) was fromSigma-Aldrich, St. Louis, Mo. The murine myeloma protein P3x63/Ag8 (P3ascitic fluid, diluted 1:500) was used as a non-reactive control murineIg for mAbs.¹⁸ In all studies, Abs were used at saturating dilutionsunless otherwise indicated.

Collection, Preparation and Treatment of RBCs.

Sickle cell patient donors had not been transfused for at least threemonths, had not experienced vaso-occlusion for three weeks, and were noton hydroxyurea. Fresh blood samples from patients homozygous forhemoglobin S and from healthy donors were collected into citrate tubes.Blood was used within less than 24 h of collection. Packed RBCs wereseparated as previously described in detail.⁵ RBCs were separated fromthe buffy coat containing leukocytes and platelet-rich plasma by gravityat 4° C. for at least 2 h. Plasma and buffy coat were removed byaspiration, and RBCs were washed four or five times in sterile PBS with1.26 mM Ca²⁺, 0.9 mM Mg²⁺ (pH 7.4). Packed RBCs were analyzed forleukocyte and platelet contamination using an Automated HematologyAnalyzer Sysmex K-1000 (Sysmex, Co., Cobe, Japan).

Aliquots of packed RBCs were treated with various reagents to affectcAMP signaling or protein phosphorylation. Sham-treated RBCs wereincubated with the same buffer and vehicle, but without the activeagent. Unless otherwise indicated, RBCs were treated at 37° C. with oneor more of the following reagents: 20 nM epinephrine (Sigma-Aldrich, St.Louis, Mo.) for 1 or 30 min; 2 mM phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX, Sigma) for 2 h; 80 μM forskolin(Sigma) for 30 min; 1 or 2 g/ml Pertussis toxin (PTx, Calbiochem, LaJolla, Calif.); 5 μM MEK1/2 inhibitor (MEKI, U0126, Calbiochem); 30 nMprotein kinase A inhibitor (PKAI) 14-22 amide (Calbiochem); 10 μMdamnacanthal (Enzo Life Sciences International, Inc., Plymouth Meeting,Pa.); or 10 μM piceatannol (Enzo Life Sciences International, Inc.) for1 h. Treated RBCs were then washed 5 times with 4 ml PBS with Ca²⁺ andMg²⁺. Normal RBCs were used as controls. Prior to adhesion assays,treated RBCs were labeled with PKH 26 red fluorescent cell linker kit(Sigma), following the manufacturer's instructions.

For in vitro adhesion assays, human SS RBCs were sham-treated withbuffer and vehicle alone or treated at 37° C. with the MEK inhibitor,U0126 (Calbiochem, La Jolla, Calif.) at 10 μM for 1 h, followed or notby treatment with 20 nM epinephrine for 1 min or 80 μM forskolin for 30min. Cells were then washed three times with 5 ml PBS with Ca²⁺ andMg²⁺. Prior to adhesion assays, washed treated SS RBCs were labeled withPKH 26 red fluorescent cell linker kit (Sigma-Aldrich, St. Louis, Mo.),following the manufacturer's instructions.

For some in vivo adhesion studies, packed SS RBCs were fluorescentlylabeled with the dye Dil (Molecular Probes Inc., Eugene, Oreg.),following the manufacturer's instructions. Dil was used in our previousin vivo studies and by other investigators, and this dye have no effecton RBC suspension viscosity and RBC survival in circulation (Unthank J Let al. Microvasc. Res. 1993; 45:193-210; Zennadi et al., Blood 2007).Cell morphology was checked by microscopy.

Western Blot.

Treated packed RBCs were lysed with hypotonic buffer (5 mM Na₂HPO₄+1 mMEDTA+0.1% NaN₃, pH 8) containing 2 mM phenylmethylsulphonylfluoride(PMSF, Sigma), phosphatase inhibitor cocktail (Sigma) and proteaseinhibitor cocktail (Sigma). Protein separation by polyacrylamide gelelectrophoresis using equal amounts of total RBC membrane ghost proteinsper lane, after correcting total protein measurements for residualhemoglobin content, and Western blot¹⁹ using the appropriate Ab werethen performed. Mouse 3T3/A31 fibroblast lysate was used as a ERK1/2positive control for immunoblots. For total ERK1/2, membranes blottedwith anti-phosphoERK Ab were stripped and reexposed to Western blottingusing anti-ERK1/2 Ab. Bands were analyzed densitometrically using ImageJsoftware downloaded from the NIH website. PhosphoERK1/2 data werenormalized according to total ERK1/2 and are presented as fold change inERK phosphorylation.

MAP Kinase Activity Assay.

Treated packed SS RBCs were lysed for 20 min at 4° C. with lysis buffer(10 mM EDTA, 20 mM Tris, 110 mM NaCl, pH 7.5) containing 2 mM PMSF, 1%Triton X-100, phosphatase inhibitor cocktail (Sigma) and proteaseinhibitor cocktail (Sigma). ERK1/2 was immunoprecipitated withanti-ERK1/2 antibody at 4° C., and immune complexes were obtained usingprotein A-agarose (Amersham Biosciences Corp., Piscataway, N.J.). ERK1/2immunocomplex was examined for ERK1/2 activity using myelin basicprotein (MBP) at 2 mg/ml (Millipore) as a substrate and ATP as aphosphate donor with equal protein amounts per assay condition. For thenegative control, an equal volume of water was substituted for ERK1/2substrate. Commercial active recombinant human ERK2 was used (Sigma) asa positive control. The reaction mixture was incubated for 20 min at 30°C., followed by protein separation and immunoblotting usinganti-phosphoMBP mAb (Millipore).

Non-radiolabeled RBC ghosts isolated from packed RBCs sham-treated, ortreated with U0126 or epinephrine for 1 or 30 min were separated by massspectrometry, and then subjected to Label-Free quantitativephosphoproteomic analysis after phosphopeptide enrichment (see below).

Reticulocyte Enrichment.

Reticulocytes were separated from mature SS RBCs using anti-transferrinreceptor mAb and goat anti-mouse IgG-coated micro-bead affinity columns(MACS, Miltenyi Biotec, Inc, Auburn, Calif.), following themanufacturer's instructions.

Flow Chamber Assays.

Graduated height flow chambers were used to quantify adhesion of RBCs toHUVECs substantially as previously described in detail.^(4, 20) In someadhesion studies, slides coated with HUVECs were treated with humanrecombinant TNF-α at 10 ng/ml for 4 hours. Slides coated with HUVECstreated or not with TNF-α were then washed three times with 20 ml HBSSwith 1.26 mM Ca²⁺, 0.9 mM Mg²⁺ (Gibco, Grand Island, N.Y.) warmedpreviously to 37° C. and then fit into a variable height flow chamber.The flow chamber was mounted on the stage of an inverted phase contrastmicroscope (Diaphot, Nikon Inc., Melville, N.Y.) connected to athermoplate (Tokai Hit Co., Ltd., Japan) set at 37° C. Cells wereobserved using a video camera (RS photometrics,) attached to themicroscope and connected to a Macintosh G4 computer. RBC (3 ml)suspended at 0.2% (vol/vol) in HBSS with Ca²⁺, Mg²⁺ were infused intothe flow chamber and allowed to adhere to the slide for 10 min withoutflow. Before exposure to flow, a minimum of three fields at each ofseven different locations along a line oriented normal to future flowwere examined for the total number of fluorescent cells. Fluid flow(HBSS with Ca²⁺, Mg²⁺) was then started using a calibrated syringe pump.After exposure to flow, the fields were again examined and the number ofadherent cells counted. The fraction of adherent cells was presented as(number of cells attached after exposure to flow)/(cells present perfield before flow). The wall shear stress was calculated as:

$\tau_{w} = \frac{6\mu\; Q}{{{wH}(x)}^{2}}$τ_(w)=wall shear stress (dyne/cm²); Q=volumetric flow rate (cm³/s); μ ismedia viscosity, w is the width of the flow channel, and H(x) is theheight of the flow chamber as a function of position along themicroscope slide. Several investigators have shown that blood flow insmall vessels may be continuous, with shear stresses of 1-2 dynes/cm²,or flow may be intermittent. Our data were obtained using bothintermittent and continuous flow conditions.

³²P Erythrocyte Labeling, Anti-ICAM-4 Immunoprecipitation and Detectionof Phosphorylation.

Packed RBCs depleted of endogenous ATP stores and ³²P-labeled aspreviously described,²¹ were incubated with phosphatase inhibitorcocktail (Sigma) in the presence or absence of MEKI U0126, PKAI, or acombination of both U0126 and PKAI, prior to 1 or 30 min treatment withepinephrine. Cells were then washed 4 times. ICAM-4 proteinimmunoprecipitation, and total and phospho-ICAM-4 detection wereperformed as previously described in detail. To further confirm that theimmunoprecipitates were specific for ICAM-4, anti-ICAM-4 mAb and thenegative control immunoglobulin P3 were used to immunoprecipitate ICAM-4from non-radiolabeled SS RBCs incubated in the presence or absence ofepinephrine. Blots were then immunostained with anti-ICAM-4 mAb.

Whole Cell cAMP Accumulation:

Whole cell cAMP accumulation was assayed to assess the functionalcapacity of the RBC β₂-ARs to stimulate the production of cAMP. Washedpacked RBCs were pre-treated with IBMX to define basal cAMPaccumulation, followed by treatment with epinephrine for 1 min or 30min, or forskolin. Samples were placed on ice, stimulation was halted,and cells fixed by the addition of 12.5 mM EDTA. Cell samples wereboiled, clarified by centrifugation and assayed for cAMP content byradioimmunoassay as described previously.²² Basal cAMP production wassubtracted from the total cAMP produced by the cells. The amounts ofcAMP were then normalized as fmol cAMP/10⁸ RBCs.

Statistical Analysis.

Data were compared using parametric analyses (GraphPad Prism 4 Software,San Diego, Calif.), including repeated and non-repeated measures ofanalysis of variance (ANOVA). One-way ANOVA analyses were followed byBonferroni corrections for multiple comparisons (multiplying the p valueby the number of comparisons). A p value <0.05 was consideredsignificant.

RBC Ghost Membrane Sample Preparation and Phosphopeptide Enrichment.

Ghosted RBCs were spun at 14,000 rpm for 15 min at 4° C. to pelletmembranes. Membrane pellets were washed with 1 mL 50 mM ammoniumbicarbonate (pH 8.0) with vortexing and were then spun at 14,000 rpm for30 min at 4° C. The supernatant was then removed and 500 μL of 50 mMammonium bicarbonate with μL 0.2% acid-labile surfactant (ALS-1) in 50mM ammonium bicarbonate (pH 8.0) was added. Samples were subjected toprobe sonication three-times for 5 sec with cooling on ice between andinsoluble material was cleared by centrifugation at 14,000 rpm for 30mins at 4° C. Samples were normalized to approximately 2 μg/μl followinga micro-Bradford assay (Pierce Bioscience), and were reduced with afinal concentration of 10 mM dithiothreitol at 80° C. for 20 min.Samples were then alkylated with a final concentration of 20 mMiodoacetamide at room temperature for 45 min and trypsin was added to afinal ratio of 1-to-50 (w/w) enzyme-to-protein and allowed to digest at37° C. for 18 hr. To remove ALS-1, samples were acidified to pH 2.0 withneat TFA, incubated at 60° C. for 2 hrs and spun at 14,000 rpm to removehydrolyzed ALS-1. Samples were either subjected directly to LC-MSanalysis or subjected to a TiO₂ based phosphopeptide enriched protocol.

To enrich for phosphorylated peptides prior to LC-MS analysis, either1,125 μg and 970 μg of total digested protein from RBC ghostsco-incubated with recombinant active ERK2 experiments and experimentsusing epinephrine-treated cells, respectively, were brought to neardryness using vacuum centrifugation and then resuspended in 200 μL of80% acetonitrile, 1% TFA, 50 mg/ml MassPrep Enhancer (pH 2.5) (WatersCorp. Milford, Mass.). Samples were loaded onto an in-house packed TiO₂spin column (Protea Biosciences) with a 562 μg or 485 μg bindingcapacity for active ERK2 treated or epinephrine treated experiments,respectively. Samples were washed twice with 200 μL 80% acetonitrile, 1%TFA, 50 mg/ml MassPrep Enhancer (pH 2.5) followed by two washes with 200μL 80% acetonitrile, 1% TFA (pH 2.5). Retained peptides were elutedtwice with 100 μL 20% acetonitrile, 5% aqueous ammonia (pH 10.0),acidified to pH 3 with neat formic acid and then brought to drynessusing vacuum centrifugation. Prior to LC-MS analysis, each sample wasresuspended in 20 pt 2% acetonitrile, 0.1% TFA, 25 mM citric acid (pH2.5).

Label-Free Quantitative Phosphoproteomic Analysis of RBC Ghost.

Chromatographic separation of phosphopeptide enriched or non-enrichedsamples was performed on a Waters NanoAquity UPLC equipped with a 1.7 μmBEH130 C₁₈ 75 μm I.D.×250 mm reversed-phase column. The mobile phaseconsisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid inacetonitrile. Five μL injections of each sample were trapped for 5 minon a 5 μm Symmetry C₁₈ 180 μm I.D.×20 mm column at 20 μl/min in 99.9% A.The analytical column was then switched in-line and the mobile phase washeld for 5 min at 5% B before applying a linear elution gradient of 5% Bto 40% B or 5% B to 30% B over 90 min at 300 nL/min for ERK2 treatedexperiments or epinephrine treated experiments, respectively. Theanalytical column was connected to fused silica PicoTip emitter (NewObjective, Cambridge, Mass.) with a 10 μm tip orifice and coupled to themass spectrometer through an electrospray interface.

MS data from each phosphopeptide enriched sample was acquired on aThermo LTQ-Orbitrap XL mass spectrometer operating in positive-ion modewith an electrospray voltage of 2.0 kV with real-time lockmasscorrection on ambient polycyclodimethylsiloxane (m/z 445.120025)enabled. The instrument was set to acquire a precursor MS scan from m/z400-2000 with r=60,000 at m/z 400 and a target AGC setting of 1e6 ions.Each sample was analyzed four-times, one of which was used foradditional qualitative identifications only and was not included in thequantitative analysis, with product ions above a threshold of 500 countswere acquired for the top 5 most intense ions in the linear ion trap.Maximum fill times were set to 1000 ms for full MS scans acquired in theOT and 250 ms for MS/MS acquired in the linear ion trap, with a CIDenergy setting of 35% and a dynamic exclusion of 60 s for previouslyfragmented precursor ions. Multistage activation (MSA) for neutrallosses of 98.0, 49.0, and 32.33 Da was enabled to enhance fragmentationof phosphorylated peptides. MS data for non-phosphopeptide enrichedsamples was acquired on a Waters Synapt HDMS operating in positive-ionmode with an electrospray voltage of 3.0 kV. Each sample was analyzedthree times in a data-independent (MS^(E)) mode of acquisition with 0.9sec cycle times alternating between low collision energy (6 V) and highcollision energy ramp (15 to 40 V). One additional data-dependent (DDA)analysis using a 0.9 sec MS scan followed by MS/MS acquisition on thetop 3 ions with charge greater than 1 was acquired to increase thenumber of qualitative identifications. MS/MS scans for each ion used anisolation window of approximately 3 Da, a maximum of 4 seconds perprecursor, and dynamic exclusion for 120 seconds within 1.2 Da.

Label-free quantitation and integration of qualitative peptideidentifications was performed using Rosetta Elucidator (v 3.3, RosettaInpharmatics, Seattle, Wash.). All raw LC-MS/MS data within anexperiment were imported and subjected to chromatographic retention timealignment using the PeakTeller® algorithm with a minimum peak time widthset to 6 s, alignment search distance set to 4 min and the refinealignment option enabled. Quantitation of all measurable signals in theprecursor MS spectra (excluding LC-MS analysis intended only foradditional qualitative identifications), was performed by Elucidator bycalculating either peak volume (area under curve) for Synapt HDMS datafiles or peak height for LTQ-Orbitrap data files.

Qualitative peptide identifications from all phosphopeptide enrichedsamples and DDA analysis of non-phosphopeptide enriched samples weremade by generating DTA files for all precursor ions, which hadassociated MS/MS spectra. DTA files were submitted to Mascot (MatrixScience, Boston, Mass.) and searched against a Homo sapien proteindatabase downloaded from SwissProt concatenated with thesequence-reversed version of each entry. MS^(E) data were independentlyprocessed within ProteinLynx Global Server 2.4 (Waters Corp) andsearchable files were then submitted to the IdentityE search engine(Waters Corp). Search tolerances of 20 ppm precursor and 0.8 Da productions were applied for LTQ-Orbitrap data and 20 ppm precursor and 0.04 Daproduct ions were applied for Synapt HDMS data files with lock-masscorrection on m/z 785.8426 (doubly-charged Glu-1-Fibrinopeptide ion)enabled. All data were searched using trypsin specificity with up to twomissed cleavages with a static modification of Carbamidomethylation(+57.0214 Da on C) and dynamic modifications of oxidation (+15.9949 Daon M). Dynamic search modifications of phosphorylation (+79.9663 Da onSTY) and of deamidation (+1.008 Da on NQ) were employed forphosphopeptide enriched sample and non-phosphopeptide enriched samples,respectively. Peptides FDR were determined by adjusting the Mascotpeptide ion score threshold to allow a 1% occurrence of peptides fromreverse protein entries for phosphopeptide enriched experiments, or byusing PeptideProphet algorithm scores which corresponded to a 2% peptidefalse discovery rate for non-phosphopeptide enriched experiments.

Database search results and spectra have been uploaded in the form ofScaffold 3 files (.sf3, Proteome Software, Inc) to the Tranche database(https://proteomecommons.org/tranche/) under the group “RBC GhostMembrane Phosphoproteome” with the following links (if a password isrequested, it is rbcphos).

Mice:

All animal experiments were carried out in accordance with protocolsapproved by the Duke University Animal Care and Use Committee. Femaleathymic homozygous nude mice (nu−/nu−) were between 8-12 weeks of age(Charles River Laboratories, Wilmington, Mass.).

Window Chamber Surgery:

General anesthesia was achieved by intra-peritoneal injection of 100mg/kg of ketamine (Abbott Laboratory, Chicago, Ill.) and 10 mg/kg ofxylazine (Bayer, Shawnee Mission, Kans.). A double-sided titanium framewindow chamber was surgically implanted into the dorsal skin fold understerile conditions using a laminar flow hood. Surgery involved carefullyremoving the epidermal and dermal layers of one side of a dorsal skinfold, exposing the blood vessels of the subcutaneous tissue adjacent tothe striated muscles of the opposing skin fold, and then securing thetwo sides of the chamber to the skin using stainless steel screws andsutures. A glass window was placed in the chamber to cover the exposedtissue and secured with a snap ring. Subsequently, animals were kept at32-34° C. until in vivo studies were performed 3 days post-surgery.

RBC Infusions and Intravital Microscopy:

Murine recombinant TNF-α was dissolved in normal saline at aconcentration of 0.1 mg/mL and mice bearing dorsal-skin window chamberimplants were given a single intraperitoneal (IP) injection of 20 g/kgTNF-α and control animals received same volume of normal saline. Threehours and 30 min following TNF-α administration, either placebo [0.4%dimethyl sulfoxide (DMSO) in normal saline] or U0126 (Cell SignalingTechnology) (2 and 0.2 mg/kg, in 0.4% and 0.04% DMSO, respectively) wasinjected intravenously via tail vein of anesthetized animals. Thirtyminutes later, labeled human SS RBCs (300 μl hematocrit (Hct) 50% in PBSwith C²⁺ and Mg²⁺) were then infused. In some experiments, animalsadministered with TNF-α were infused 4 hours later with washed SS RBCssham-treated or treated with 10 μM U0126 or 10 μM RDEA119 for 1 hour.Animals were placed on the stage of an Axoplan microscope (Carl Zeiss,Thornwood, N.Y.); temperature was maintained at 37° C. using athermostatically controlled heating pad. RBC adhesion and blood flowdynamics were observed in subdermal vessels for at least 30 minutesusing 20× and 10× magnifications. Microcirculatory events and celladhesion were simultaneously recorded using a Trinitron Color videomonitor (PVM-1353 MD, Sony) and JVC videocassette recorder (BR-S3784,VCR King, Durham, N.C.) connected to a digital video camera C2400(Hamamatsu Photonics K.K., Japan). Arterioles were distinguished fromvenules based on: 1) observation of divergent flow as opposed toconvergent flow; 2) birefringent appearance of vessel walls usingtransillumination, which is characteristic of arteriolar vascular smoothmuscle; and 3) relatively straight vessel trajectory without evidence oftortuosity. Cell adherence was quantitated by considering cells attachedto the vessel walls and immobile for 1 minute. The percentage of thelength of vessels with diameters ≦25 μm or >25 μm, occupied by SS RBCswas quantified as: % venular length occupied by SS RBCs=length of vesselwall with adherent cells/total length of the vessel segmentsanalyzed×100.

Example 2 ERK1/2 is Present in Mature RBCs and Undergoes Activation byEpinephrine in SS but not Normal RBCs

Recently, our preliminary data showed that ERK1/2 can be found bound tothe RBC plasma membrane. The cAMP/PKA pathway is known to both activateSS RBCs to adhere abnormally to endothelial cells (ECs)⁴ and modulatethe MAPK/ERK cascade. Given the importance of abnormal SS RBC adherencein SCD pathophysiology, we investigated the possibility that ERKactivity is conserved in SS RBCs and inducible by epinephrine. RBCghosts consisting of membrane fragments prepared from SS and normal (AA)RBCs were first analyzed to confirm the presence of ERK1/2 and MEK1/2,the upstream kinase of ERK1/2 activation. MEK1/2 was abundant in both SSand AA RBCs, while ERK1/2 was expressed at higher levels in SS vs AARBCs (p<0.05, FIGS. 1A and B). Since ERK1/2 is also well expressed byplatelets and leukocytes, we examined our RBC suspensions forcontamination by other blood cells. Our SS RBC preparations(0.13±0.01×10/ml RBCs) showed no contamination by platelets, but a verylow level of contamination by leukocytes (0.2±0.06×10³/ml) was sometimesdetected. However, when similar numbers of isolated sickle cell patientleukocytes were examined for the presence of ERK1/2, no detectablesignal was observed (data not shown), making it apparent that theobserved ERK signal was in fact derived from SS RBCs.

Our data also indicated that ERK1/2 is phosphorylated at baseline in SSRBCs, and epinephrine at a physiologic “stress” dose (20 nM)²⁹ promoteda 2.1±0.1-fold increase in ERK phosphorylation within 1 minute (n=3;p<0.001) (FIGS. 1C and D). Incubation of SS RBCs with the MEKI U0126,which specifically inhibits MEK1/2, prior to epinephrine treatment,significantly inhibited the effect of epinephrine on ERK1/2phosphorylation (p<0.001) (Figures IC and D). Because our previous dataindicated that the degree of adhesive response to epinephrinestimulation varied from patient to patient,⁴ the effect of epinephrineon ERK phosphorylation was measured in samples obtained from a largergroup of patients (n=19). Although a statistically significant increase(2±0.17-fold) in ERK phosphorylation above basal levels was observed(P<0.05), SS RBCs from only 40% of patients exhibited more than 1.5-foldelevation in ERK phosphorylation by epinephrine. These patients wereclassified as responders. This data suggests that not all SCD patientsare susceptible to epinephrine-stimulated increased ERK phosphorylation.In contrast, ERK1/2 was never found phosphorylated in AA RBCs and failedto undergo phosphorylation by epinephrine (FIG. 1C). In contrast, ERK1/2was never found phosphorylated in normal (AA) RBCs at baseline and alsofailed to undergo phosphorylation after epinephrine stimulation (FIG.1C).

To further confirm that ERK1/2 preserved its activity in SS RBCs andthat phosphorylation was indeed an indicator of ERK activation, we usedthe ERK specific substrate, myelin basic protein (MBP), to test theactivity of ERK1/2 isolated from both sham-treated andepinephrine-treated SS RBCs, in the presence of inhibitors of PKA, PKC,Ca²⁺/calmodulin-dependent kinase and p34^(cdc2) kinase to preventnonspecific phosphorylation of MBP by these enzymes.²⁴ ERK1/2immunoprecipitated from sham-treated SS RBCs was capable ofphosphorylating MBP to some extent, while MBP phosphorylation by ERK1/2immunoprecipitated from epinephrine-treated 55 RBCs increased2.1±0.3-fold compared to MPB phosphorylation induced by ERK1/2 isolatedfrom sham-treated cells (n=4; p=0.0286) (FIGS. 1E and F). These dataindicate that ERK1/2 can be already somewhat activated in SS RBCs andthat epinephrine can augment its activity.

Example 3 ERK1/2 in SS RBCs Acts Downstream of the cAMP/PKA SignalingPathway

We found that treatment of SS RBCs with forskolin, which directlyactivates AC to produce cAMP, promoted increased ERK1/2 phosphorylation,which was in turn prevented by MEKI U0126, suggesting that cAMP isneeded for ERK activation in SS RBCs (FIG. 2A).

To determine the role of PKA in ERK phosphorylation, we used thePKA-specific inhibitor (PKAI), 14-22 amide. Treatment of SS RBCs withthe PKAI, 30 nM 14-22 amide, at a concentration known to promote optimalinhibition of PKA in SS RBCs, did not significantly decrease basal ERKphosphorylation in these sickle cells (FIG. 2B). However, PKAIcompletely blocked the effect of epinephrine on ERK phosphorylation(p<0.01, n=3). Pre-treatment of SS RBCs with a combination of PKAI andMEKI U0126 also completely blocked ERK phosphorylation in response toepinephrine stimulation (p<0.01) (FIG. 2B). Together these data suggestthat ERK1/2 activation in SS RBCs is dependent on the cAMP/PKA pathway.

In some instances, β₂AR activation employs a Gα_(i) (or Gα_(o)) pathwayto stimulate ERK activity.⁷ We investigated whether epinephrinestimulated SS RBC β₂ARs mediated ERK activation also involved theGα_(i), using Pertussis toxin (PTx), which inhibits Gα_(i)-signaling.Inhibition of Gα_(i) with 1 or 2 μg/ml PTx alone significantly increasedbasal phosphorylation of ERK1/2 by 2.04±0.1- and 2.53±0.11-fold,respectively and combining PTx with epinephrine had no additional effect(FIG. 2C, p<0.001). These results suggest that increased ERK1/2phosphorylation in SCD patient samples tested is negatively affected byGα_(i) activation, or due to the direct actions of PTx.

Because direct or indirect involvement of cytoplasmic tyrosine kinasesin activation of MAP kinase cascades has also beendemonstrated,^(25, 26) we evaluated the contribution of tyrosinekinase-induced signaling to RBC ERK1/2 phosphorylation. ERK1/2 wasphosphorylated at baseline in sham-treated SS RBCs (FIG. 2D). Treatmentwith 2 μg/ml PTx markedly increased ERK1/2 phosphorylation.Damnacanthal, a highly potent and selective inhibitor of the tyrosinekinase p56^(syk,27) did not abrogate ERK phosphorylation in response toPTx (FIG. 2D). However, piceatannol, which preferentially inhibits thetyrosine kinase p72^(syk) vs p56^(lyss), completely blocked the effectof PTx on ERK phosphorylation. Once more, U0126 blocked the effect ofPTx on ERK1/2 phosphorylation. These data suggest that thepiceatannol-sensitive tyrosine kinase p72^(syk) also plays a role in SSRBC ERK1/2 activation.

To determine if ERK1/2 is active only in the youngest cell population(reticulocytes), reticulocyte-enriched and -depleted (mature) SS RBCswere analyzed for kinase phosphorylation. Flow cytometric analysisshowed that up to 15% of unseparated SS RBCs expressed the transferrinreceptor, a reticulocyte marker. After separation, more than 95% of thereticulocyte-enriched cells expressed the transferrin receptor, whilethe reticulocyte-depleted population reacted with the anti-transferrinreceptor antibody no more strongly than with the negative controlimmunoglobulin (data not shown). ERK1/2 was strongly phosphorylated inboth reticulocyte-enriched and reticulocyte-depleted cells (n=2) (FIG.2E), suggesting that ERK activity is preserved in both reticulocytes andmature SS RBCs.

Example 4 ERK1/2 is Involved in SS RBC Adhesion to Endothelial Cells

Since the pharmacological agents epinephrine and forskolin modulate bothSS RBC adhesion to ECs and ERK activation, we determined thecontribution of MEK/ERK signaling to RBC adhesion. Epinephrinesignificantly up-regulated SS RBC adhesion to HUVECs at a shear stressof 2 dynes/cm² in intermittent flow condition assays (p<0.001) (FIG.3A). However, U0126 completely inhibited the effect of epinephrine on SSRBC adhesion (p<0.001). Treatment of SS RBCs with U0126 alone alsoblocked SS RBC adhesion to HUVECs (91±4.6% inhibition) when compared toadhesion of sham-treated SS RBCs (p<0.01).

Forskolin also enhanced SS RBC adhesion to HUVECs at a shear stress of 2dynes/cm² (p<0.001, n=3) (FIG. 3B), and this effect was blocked by U0126(83±4% inhibition, compared to increased adhesion by forskolin alone;p<0.01). This suggests that the MEK/ERK pathway contributes toup-regulation of SS RBC adhesive function to ECs.

Example 5 ERK Signaling is Implicated in Phosphorylation of the RBCAdhesion Receptor ICAM-4 (Landsteiner-Wiener Blood Group Antigen, LW)

We further explored the possibility that the ERK signaling pathway isinvolved in ICAM-4 (LW) phosphorylation, which mediates adhesion viabinding to endothelial αvβ3 integrin.⁴ The ICAM-4 protein possesses onlyone serine, one tyrosine and no threonine within the 12 amino acids ofits cytoplasmic tail, and it does not contain a typical PKA targetconsensus motif. Nevertheless, up-regulation of SS RBC adhesion tonon-activated ECs requires serine phosphorylation of the ICAM-4receptor.⁴ PhosphorImager analysis of immunoprecipitated³²P-radiolabeled ICAM-4 and negative control immune complexes showedthat ICAM-4 of non-stimulated SS RBCs (FIG. 3C, lane 1) is modestlyphosphorylated as previously shown. Treatment of 55 RBCs with serinephosphatase inhibitors (SPI) (lane 2) increased ICAM-4 phosphorylationby 3.7±0.46-fold (p<0.05, n=3), suggesting that increased ICAM-4phosphorylation is a result of serine phosphorylation, as tyrosinephosphatase inhibitors were not present. These were similar to theeffects of epinephrine, although SPI-stimulation induced a significantincrease (2.62±0.6-fold) in ICAM-4 phosphorylation above baseline in alarger group of patients (n=8) (P<0.05), only half of all SS RBC samplesexhibited a 2-fold elevation in ICAM-4 phosphorylation in response toSPI. Epinephrine in the presence of SPI had a stronger effect on ICAM-4phosphorylation (7.4±1.07-fold increase over sham-treated SS RBCs;p<0.001) (lane 3). Treatment of SS RBCs with either the PKAI or U0126(lanes 4 and 5, respectively) significantly decreased the combinedeffect of epinephrine and SPI on ICAM-4 phosphorylation compared tocells treated with epinephrine alone (p<0.001) (lane 3). Treatment of 55RBCs with both PKAI and MEKI completely blocked epinephrine and SPI fromup-regulating phosphorylation of ICAM-4 (p<0.001) (FIG. 3C, lane 6).

Immunoblots of ³²P-radiolabeled ICAM-4 immunoprecipitates fromstimulated and non-stimulated SS RBCs (FIG. 3C) indicated that a similaramount of ICAM-4 was immunoprecipitated from these cells. Controlimmunoblots of immunoprecipitated ICAM-4 and the negative controlcomplexes immunoprecipitated with P3 from stimulated and non-stimulatedSS RBCs are shown in FIG. 3D.

To define whether ICAM-4 is a substrate for ERK, we used non-treatedpacked normal RBCs as a source of ICAM-4, since ERK is inactive in thesecells (FIG. 1) and ICAM-4 is not phosphorylated at baseline.⁴ Exposureof immunoprecipitated ICAM-4 to active recombinant ERK2 did not causeICAM-4 phosphorylation, indicating that ICAM-4 is not a substrate forERK (data not shown). Together, our data demonstrate that ICAM-4 in SSRBCs undergoes serine phosphorylation by a yet unknown kinase, and thisprocess is PKA and MEK/ERK1/2 dependent.

Example 6 SS RBC Adhesion is Strictly Related to the Inception of ERKActivation

Epinephrine significantly increased SS RBC adhesion to HUVECs under bothintermittent and constant flow conditions after 1 min exposure (p<0.001for each), while adhesion decreased after 30 min cell exposure toepinephrine (mean decrease of all samples=56±1.5% and 73±4.7% forintermittent and constant flow conditions, respectively; p<0.001 foreach) (FIGS. 4A and B). In contrast, epinephrine treatment for either 1or 30 min had minimal effect on normal RBC adhesion to HUVECs undereither intermittent or constant flow conditions (FIGS. 4A and B).

We also examined the effect of exposure time of SS RBCs to epinephrineon cAMP production, which appears to act upstream of ERK1/2. Basal cAMPin normal RBCs from healthy donors was significantly lower than basalcAMP in SS RBCs (p=0.0187) (FIG. 4C). In about 50% of the samplesexamined (n=19), incubation of SS RBCs with epinephrine for 1 minresulted in accumulation of high levels of intracellular cAMP comparableto the levels of cAMP induced by forskolin treatment for 30 min.Although the cAMP response to epinephrine (1 min exposure time) variedamong patients, as previously described,²⁸ cAMP levels uniformlydeclined with 30 min exposure time of SS RBCs to epinephrine (p<0.05)(FIG. 4C). Epinephrine exposure for 1 min had lower effect on cAMPaccumulation in normal RBCs (n=12) than in SS RBCs (FIG. 4C) aspreviously shown.

Additionally, while a 1 min exposure of SS RBCs to epinephrine markedlyincreased ERK phosphorylation (p<0.01 for epinephrine-treated for 1 minvs sham-treated), ERK phosphorylation decreased after a 30 min exposure(p<0.01 for epinephrine-treated for 1 min vs 30 min) to levels observedin sham-treated cells (p>0.05 for epinephrine-treated for 30 min vssham-treated) (FIGS. 4D and E).

ICAM-4 phosphorylation also decreased with longer exposure time (30 minvs 1 min) of SS RBCs to epinephrine (FIG. 4F). PhosphorImager analysisof immunoprecipitated ³²P-radiolabeled ICAM-4 and negative controlimmune complexes showed that treatment of SS RBCs with epinephrine for 1min in the presence of SPI (lane 2) enhanced ICAM-4 phosphorylation by1.84±0.15-fold over sham-treated cells (lane 4, p<0.01). Thirty minutesexposure of SS RBCs to epinephrine in the presence of SPI (lane 3)significantly diminished the effect of epinephrine and SPI on ICAM-4phosphorylation compared to cells treated with epinephrine for 1 min(lane 2, p<0.05). Altogether, these data indicate that exposure time toepinephrine influences all these downstream effects—SS RBC adhesion,cAMP levels and phosphorylation of both ERK1/2 and ICAM-4—in a parallelfashion, suggesting that the time course of up-regulation ofICAM-4-mediated SS RBC adhesion is closely associated to the extent ofERK1/2 activation.

To identify potential proteins involved in regulation of the ERKpathway, a label-free quantitative phosphoproteomics analysis of RBCghosts isolated from SS and normal RBCs treated with epinephrine for 1and 30 min was undertaken. SS RBCs treated with epinephrine for 30 minshowed a dramatic decrease in phosphorylation of serine 310 withinadenylate cyclase-associated protein 1 (CAP1) compared to cellsstimulated with epinephrine for 1 min (−4.3-fold, p=1.54×10⁻⁵) (FIG.4G). Conversely, AA RBCs exposed to epinephrine for 30 min showed anenhancement in phosphorylation of the CAP1 serine vs 1 min epinephrineexposure (+1.4-fold, p=0.008). Threonine 307 within CAP1 also underwenta smaller yet statistically significant decrease in phosphorylation inSS RBCs exposed to epinephrine for 30 min vs 1 min epinephrine exposure(−1.4-fold, p=4.92×10⁻⁷). Our data indicate that exposure to epinephrinefor a prolonged period of time negatively affects phosphorylation ofCAP1 in SS but not in AA RBCs. Adenylate cyclase-associated proteins(CAPs) are known to regulate AC activation to increase cAMP levels underspecific environmental conditions. We therefore suggest that a decreasein CAP1 phosphorylation in SS RBCs might down-regulate AC activity inthese cells, negatively affecting signaling downstream of ERK.

Example 7 ERK Signaling Pathway is Implicated in Phosphorylation ofProtein 4.1

A label-free quantitative phosphoproteomics analysis was also performedto identify additional putative downstream targets of ERK by addingrecombinant active ERK2 to RBC ghosts isolated from SS and normal RBCs.Because endogenous ERK is active at baseline in SS but not normal RBCs(FIG. 1), SS RBCs were treated with U0126 prior to incubation of theghosts with recombinant ERK2. We found that phosphorylation of protein4.1 was induced in the presence of recombinant ERK2. Treatment of SSRBCs with U0126 resulted in a significant decrease (−1.7-fold,p=1.01×10⁻⁶) of a Ser540/Ser542 doubly phosphorylated peptide withinprotein 4.1 (FIG. 5). Addition of recombinant ERK2 to the U0126-treatedSS RBC ghosts increased the abundance of this phosphopeptide (+1.7-fold,p=8.06×10⁻¹⁴) back to levels observed in untreated SS RBCs, indicatingthe specificity of ERK2 as the upstream kinase. As expected, treatmentof AA RBCs with U0126 did not induce a decrease of this doublyphosphorylated peptide within protein 4.1, since endogenous ERK isinactive in these cells. However, the complementary trend for thisphosphorylated peptide was observed upon the addition of recombinantERK2 to untreated and U0126-treated AA RBC ghosts, for which an increaseof 2.1-fold (p=1.5×10⁻¹² for untreated AA RBCs vs untreated AARBCs+ERK2) and 1.7-fold (p=4.6×10⁻⁶ for U0126-treated AA RBCs vsU0126-treated AA RBCs+ERK2) were measured, respectively.

To confirm that the measured changes in phosphorylated peptide levelswere not due to a difference in protein level between these treatmentconditions, a non-phosphopeptide enriched proteomic analysis of AA RBCghosts and AA RBC ghosts co-incubated with recombinant ERK2 wasperformed, and confirmed that protein 4.1 levels were similar betweenthe two conditions (FIG. 7A). This indicates that the observed changesin phosphopeptide abundance results from upstream kinase activity.Similar results were obtained when SS RBC ghosts and SS RBC ghostsco-incubated with recombinant ERK2 were analyzed (FIG. 78).

Collectively, these data further strengthen our findings that ERK isactive in SS RBCs, and suggest that activation of the ERK cascadeinduces phosphorylation of the cytoskeletal protein 4.1.

Example 8 Recombinant Active ERK2 Phosphorylates the ERK Consensus Motifon Dematin and Adducins α and β

To identify ERK substrates in RBCs, all phosphopeptide sequences withinthe dataset identified when active recombinant ERK2 was added to RBCghosts were searched for the known ERK consensus motif, [PV]×[pST]P.Adducin-α and -β, and dematin, contained nine, seven and one uniquephosphorylated peptides, respectively, with phosphorylation of residueswithin the ERK consensus motif. Only the statistically significantphosphopeptides with fold-changes of >1.5 are listed in Table 1. Thesepeptides underwent a significant increase in phosphorylation in AA RBCswhen recombinant ERK2 was added to the ghosts, while a decrease inphosphorylation of these peptides was observed in U0126-pretreated SSRBCs (Table 1). This suggests that the cytoskeletal proteins adducins αand β and dematin are substrates for ERK in RBCs.

Table 1.

Motif Specific Phosphorylation by active recombinant ERK2. Fold changesin phosphorylation for peptides containing the ERK consensus motif[PV]×[pST]P were presented. Phosphorylation is up-regulated in normalRBCs (AA) with addition of active ERK2 and down-regulated in SS RBCs(SS) with addition of the MEK inhibitor U0126.

TABLE 1 Motif Specific Phosphorylation by Active Recombinant ERKZProtein Modified Peptide Sequence AA + ERK2 P SS + U0126 P Description(SEQ ID NO:) vs. AA value vs. SS value α-adducin [pS]PG[pS]PVGEGTGSPPK −1.71 0.035 1.47 0.122 (SEQ ID NO: 2) α-adducinEEEAHRPP[pS]PTEAPTEASPEPAPDPAPVAEE −1.69 0.025 1.60 0.074AAPSAVEEGAAADPG[pS]DGSPGK (SEQ ID NO: 3) β-adducinETAPEEPG[pS]PAK[p5]APA[p5]PVQSPAK −2.36 4.21E− 1.91 3.87E−(SEQ ID NO: 4) 06 09 β-adducin ETAPEEPGSPAK[pS]APA[pS]PVQSPAK −1.750.020 1.82 0.018 (SEQ ID NO: 5) β-adducin ETAPEEPG[pS]PAK[pS]APASPVQSPAK−1.68 0.037  1.68 0.010 (SEQ ID NO: 6) Dematin [pS]TSPPP[pS]PEVWADSR−1.76 9.46E− 1.48 4.98E− (SEQ ID NO: 7) 09 04

Fold changes in phosphorylation for peptides containing the ERKconsensus motif [PV]×[pST]P were presented. Phosphorylation isup-regulated in normal RBCs (AA) with addition of active ERK2 anddown-regulated in SS RBCs (SS) with addition of the MEK inhibitor U0126.

Example 9 ERK1/2 is Involved in SS RBC Adhesion to Activated-EndothelialCells

We have previously shown that both pharmacological agents epinephrineand forskolin upregulate SS RBC adhesion to non-activated endothelialcells via the MEK/ERK signaling pathway (Zennadi et al., Blood 2012).Treatment of SS RBCs with the MEK inhibitor U0126 alone alsosignificantly blocked SS RBC adhesion to non-activated HUVECs (91±4.6%inhibition) when compared to adhesion of sham-treated SS RBCs (p<0.01).These data suggest that increased SS RBC adhesion to non-activatedendothelial cells requires activation of RBC adhesion molecules viastimulation of the MEK/ERK pathway.

Since inflammatory molecules are commonly augmented in sickle celldisease patients, and because Kaul et al. (Blood 2000; 95:368-374) hasreported that human SS RBCs adhered to cytokine-stimulated postcapillaryendothelium in the absence of plasma, we asked whether ERK innon-stimulated SS RBCs also contributes to SS RBC adhesion toTNF-α-activated HUVECs. Treatment of HUVECs with TNF-α resulted in asignificant increase in SS RBC adhesion by 2.6-fold at a shear stress of2 dynes/cm² in intermittent flow conditions when compared to adhesion ofSS RBCs to non-activated HUVECs, where less than 30% of the cells wereable to adhere (p<0.001) (FIG. 8A). However, pre-treatment of SS RBCswith 10 μM U0126 significantly reduced adhesion of SS RBCs toTNF-α-activated HUVECs and only 18±5% of the cells adhered compared toadhesion of SS RBCs to non-activated HUVECs (p<0.001). These resultssuggest that ERK is active in SS RBCs without prior cell-stimulation andis also involved in adhesion to activated-endothelial cells.

To confirm these initial data, we performed a similar experiment usingthree other inhibitors of MEK, RDEA119, AZD6244 and GSK1120212. Theseinhibitors were selected based on their good tolerability in long-termhuman therapeutic studies. Treatment of SS RBCs with RDEA119, AZD6244and GSK1120212 (FIG. 8B) significantly abolished SS RBC adhesion toTNF-α-activated endothelial cells to levels below baseline adhesion ofSS RBCs to non-activated HUVECs similar to the results shown for U0126above. These data suggest that ERK signaling is involved in abnormal RBCadhesive interactions with activated-endothelium, and does NOT requireactivation of the RBCs to mediate RBC adhesion.

Example 10 ERK Contributes to SS RBC Adhesion to Vascular Endotheliumand Vaso-Occlusion In Vivo

The following experiment was designed to determine whether the MEKinhibitor U0126 can be used as a preventive agent of SS RBC adhesion toactivated endothelium and vaso-occlusion. Human SS RBC preparationsshowed unmeasurable (0 cells/μl) leukocytes or platelets, making itunlikely that human leukocytes and platelets could participate in SS RBCadhesion and vaso-occlusion in our model.

All human SS RBCs were fluorescently labeled, then washed prior toinfusion into animals for observation within native intact vessels.Infusion of SS RBCs to mice injected with TNF-α resulted in marked SSRBC adhesion to vessels, predominantly in postcapillary venules, withintermittent occlusion of vessels and permanent blockage of some vesselsegments (FIG. 9A). Vaso-occlusion occurred most frequently wherevessels curved and at junctions, although it was also observed instraight non-junctional venular segments. SS RBC adhesion was evengreater when mice were infused with vehicle (placebo, 0.4% DMSO innormal saline) 30 min prior to RBC infusion (FIG. 9B) than that observedwithout vehicle infusion (FIG. 9A). SS RBC adhesion to venularendothelium occurred in less than 5 min after RBC infusion, and led tothe permanent obstruction of small diameter vessels (FIG. 9B). SS RBCadhesion was also observed in larger vessels. Vaso-occlusion alsooccurred most frequently where vessels curved and at junctions, althoughit was also observed in straight non-junctional venular segments. Incontrast, SS RBCs showed very little adhesion to vascular endotheliumand adhered only occasionally to small postcapillary venules whenanimals were infused with 2 mg/kg U0126 in 0.4% DMSO 30 min prior to RBCinfusion with no apparent vaso-occlusion (FIG. 9C). Similarly, whenanimals were infused with 0.2 mg/kg U0126 in 0.04% DMSO in saline 30minutes prior to RBC infusion no apparent vaso-occlusion was observed(FIG. 9D). The effect of U0126 on % venular length occupied by SS RBCswas quantified. The MEK inhibitor U0126 at 0.2 and 2 mg/kg induced asignificant decrease in percentage venular length occupied by SS RBCsfor vessels ≦25 μm in diameter (p<0.001) (FIG. 9E). However, when vesseldiameter was >25 μm, U0126 at 0.2 mg/kg did not significantly reduce thepercentage venular length occupied by SSRBCs. These data stronglysuggest that ERK is implicated in SS RBC adhesion to poscapillaryvenules promoting vaso-occlusion, and that the MEK inhibitor U0126 isable to down-regulate adhesion of subsequently infused SS RBCs andprevent precipitation of occlusion of enflamed vessels when provided inthe circulation. These data also suggest that MEK inhibitors may be ableto reduce inflammation.

To further these studies, we tested whether the MEK inhibitor preventsSS RBC-induced vasoocclusion via at least its effect on SS RBCs. SS RBCswere treated with the MEK inhibitors U0126 and RDEA119 ex vivo prior toadministration to the mice. Intravital microscopy studies showed thatinfusion of sham-treated human SS RBCs to nude mice treated with TNF-α(n=5), showed marked adhesion in inflammed venules and induced occlusionof small diameter (9-25 μm) vessels (n=5). SSRBC adhesion was alsoobserved in much larger vessels (up to 100 μm in diameter), indicatingthat human SSRBC-induced vasoocclusion was not a result of trapping ofhuman SSRBCs in vessels with diameters ≦8 μm, since the size of humanRBC is 8 μm in diameter (FIG. 10A). However, inhibition of MEK/ERK inhuman SSRBCs with U0126 ex-vivo prior to RBC infusion to animals treatedwith TNF-α (n=5), dramatically decreased human SSRBC adhesion andprevented vessel obstruction (FIG. 10B). Similarly, treatment of humanSS RBCs with RDEA119 (n=5) also dramatically decreased human SS RBCadhesion and prevented vessel obstruction (FIG. 10C). The Effect ofU0126 and RDEA119 on % venular length occupied by SS RBCs, wasquantified. The percentage of venular length occupied by bothU0126-treated SS RBCs and RDEA119-treated SS RBCs significantlydecreased for vessels <25 μm in diameter (p<0.001 compared to sham) andvessels with a diameter >25 μm (p<0.05 compared to sham) (FIG. 10D).These data suggest that MEK inhibition with U0126 and RDEA119 improvedSS RBC circulatory behavior due to amelioration of SS RBC adhesivefunction. Thus, our data suggest that ERK and its mechanism of actioncould represent a novel target for the treatment of SCD pathophysiology.

Example 11 Incubation of Polymorphonuclear Cells with SSRBCs Results inActivation of the Polymorphonuclear Cells

To further analyze the effect of ERK activation on SSRBCs, we treatedSSRBCs with 20 nM epinephrine alone for 1 min or after pre-incubationwith 10 mM U0126, RDEA119, AZD6244 or GSK1120212 for 1 hour, followed bytreatment with 20 nM epinephrine for 1 min. The cells were then washedprior to admixture with fluorescently-labeled native polymorphonuclearcells (PMNs) obtained from healthy donors at a RBC:PMN ratio of 10:1.After co-incubation for 30 minutes at 37° C., cells mixtures wereassayed for their ability to adhere to non-activated HUVECs. Since theonly cell population visualized was red-fluorescence labeled normalPNMs, the quantitation of adherent PMNs did not include non-labeled SSRBCs or any remaining non-labeled leukocytes from SCD patients. Our datashow that co-incubation of epinephrine-activated SS RBCs with naivePMNs, resulted in significant activation of PMN adhesion tonon-activated endothelial cells compared to adhesion of native normalPMNs not co-incubated with SS RBCs (FIG. 11). However, blocking ERKactivity in activated-SSRBCs with MEK inhibitors U0126, RDEA119, AZD6244and GSK1120212 significantly decreased the ability of activated-SS RBCsto promote neutrophil adhesion (FIG. 11). These data suggest that ERKsignaling is involved in activation of SS RBC adhesion receptorsinvolved in interactions with the endothelium and leukocytes.Downregulation of RBC adhesion receptor activity by targeting ERK maynot only decrease SS RBC adhesion to the endothelium but also reduceRBC-stimulated leukocyte activation.

Example 12 Phosphoproteomic Profiling of RBC Membranes

However, sickle cell adhesion and vaso-occlusion alone do not accountfor the pathophysiology of SCD. Subsequent changes in red cell membranestructure and function and disordered cell volume control may also playan important role. Therefore, we investigated the ERK1/2 mediated RBCprotein phosphorylation in SS RBC plasma membrane as compared to AA RBCplasma membrane.

Collection, Preparation and Treatment of RBCs.

Human SCD patients homozygous for hemoglobin S were not transfused forat least three months, had not experienced vaso-occlusion for threeweeks, and were not on hydroxyurea. Blood samples from SCD patients andhealthy donors collected into citrate tubes, were used within less than24 h of collection. Packed RBCs were separated as previously describedin detail.⁶⁶ Packed RBCs were analyzed for leukocyte and plateletcontamination using an Automated Hematology Analyzer K-1000 (Sysmex,Japan). For proteomics studies, aliquots of packed RBCs were treated at37° C. with 10 M MEK1/2 inhibitor U0126 (Calbiochem, La Jolla, Calif.)for 1 hour. Sham-treated RBCs were incubated with the same buffer andvehicle, but without the active agent. Normal RBCs were used ascontrols.

MAP Kinase Activity Assay.

Treated packed normal and SS RBCs were lysed at 4° C. with lysis buffer(10 mM EDTA, 20 mM Tris, 110 mM NaCl, pH 7.5) containing 2 mM PMSF, 1%Triton X-100, phosphatase inhibitor cocktail (Sigma) and proteaseinhibitor cocktail (Sigma-Aldrich, St. Louis, Mo.). RBC membrane ghostswere then incubated with or without recombinant active human ERK2(sigma) at 8.2 μg/ml with a specific activity of 700 nmole/min/mg, inthe presence of inhibitors of PKA, PKC, Ca²⁺/calmodulin-dependent kinaseand p34^(cdc2) kinase to prevent nonspecific protein phosphorylation bythese enzymes,⁵⁷ and with ATP as a phosphate donor with equal membraneghost protein amounts per assay condition. For the negative control, anequal volume of water was substituted for ATP. The reaction mixture wasincubated for 20 min at 30° C. To stop the enzymatic reaction sampleswere placed on ice.

RBC Membrane Ghost Preparation and Phosphopeptide Enrichment.

Non-radiolabeled RBC membrane ghosts isolated from packed RBCssham-treated or treated with U0126 and incubated with or withoutrecombinant ERK2, were spun at 14,000 rpm for 15 min at 4° C. to pelletmembranes. Membrane pellets were washed with 1 mL 50 mM ammoniumbicarbonate (pH 8.0) with vortexing and were then spun at 14,000 rpm for30 min at 4° C. The supernatant was then removed and 500 μL of 50 mMammonium bicarbonate with μL 0.2% acid-labile surfactant (ALS-1) in 50mM ammonium bicarbonate (pH 8.0) was added. Samples were subjected toprobe sonication three-times for 5 sec with cooling on ice between andinsoluble material was cleared by centrifugation at 14,000 rpm for 30min at 4° C. Samples were normalized to approximately 2 μg/μl followinga micro-Bradford assay (Pierce Biotechnology, Inc), and were reducedwith a final concentration of 10 mM dithiothreitol at 80° C. for 20 min.Samples were then alkylated with a final concentration of 20 mMiodoacetamide at room temperature for 45 min and trypsin was added to afinal ratio of 1-to-50 (w/w) enzyme-to-protein and allowed to digest at37° C. for 18 hr. To remove ALS-1, samples were acidified to pH 2.0 withneat TFA, incubated at 60° C. for 2 hrs and spun at 14,000 rpm to removehydrolyzed ALS-1. Samples were either subjected to LC-MS analysisfollowing a 10× dilution into mobile phase A or subjected to a TiO₂based phosphopeptide enriched protocol.

To enrich for phosphorylated peptides prior to LC-MS analysis, 1,125 μgof total digested protein from RBC ghosts were brought to near drynessusing vacuum centrifugation and then resuspended in 200 μL of 80%acetonitrile, 1% TFA, 50 mg/ml MassPrep Enhancer (pH 2.5) (Waters Corp.,Milford, Mass.). Samples were loaded onto an in-house packed TiO₂ spincolumn (Protea Biosciences) with a 562 μg binding capacitypre-equilibrated with 80% acetonitrile, 1% TFA (pH 2.5). For allloading, washing, and elution steps, the centrifuge was set to achieve aflow rate of no faster than 100 μL/min. Samples were washed twice with200 μL 80% acetonitrile, 1% TFA, 50 mg/ml MassPrep Enhancer (pH 2.5)followed by two washes with 200 μL 80% acetonitrile, 1% TFA (pH 2.5).Retained peptides were eluted twice with 100 μL 20% acetonitrile, 5%aqueous ammonia (pH 10.0), acidified to pH 3 with neat formic acid andthen brought to dryness using vacuum centrifugation. Prior to LC-MSanalysis, each sample was resuspended in 20 μL 2% acetonitrile, 0.1%TFA, 25 mM citric acid (pH 2.5).

Label-Free Quantitative Proteomic Analysis of RBC Membrane Ghosts.

Chromatographic separation of phosphopeptide enriched or non-enrichedsamples was performed on a Waters NanoAquity UPLC equipped with a 1.7 μmBEH130 C₁₈ 75 μm I.D.×250 mm reversed-phase column. The mobile phaseconsisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid inacetonitrile. Five μL injections of each sample were trapped for 5 minon a 5 μm Symmetry C₁₈ 180 μm I.D.×20 mm column at 20 μl/min in 99.9% A.The analytical column was then switched in-line and the mobile phase washeld for 5 min at 5% B before applying a linear elution gradient of 5% Bto 40% B over 90 min at 300 nL/min. The analytical column was connectedto fused silica PicoTip emitter (New Objective, Cambridge, Mass.) with a10 μm tip orifice and coupled to the mass spectrometer through anelectrospray interface.

MS data from each phosphopeptide enriched sample was acquired on aThermo LTQ-Orbitrap XL mass spectrometer operating in positive-ion modewith an electrospray voltage of 2.0 kV with real-time lockmasscorrection on ambient polycyclodimethylsiloxane (m/z 445.120025)enabled. The instrument was set to acquire a precursor MS scan from m/z400-2000 with r=60,000 at m/z 400 and a target AGC setting of 1e6 ions.Each sample was analyzed four-times, one of which acquired MS/MS spectrain the ion-trap for the top 10 most abundant precursor ions and was usedfor additional qualitative identifications only. All other quantitativeanalysis acquired MS/MS spectra in the ion-trap for the top 5 mostabundant precursor ions above a threshold of 500 counts. Maximum filltimes were set to 1000 ms for full MS scans acquired in the OT and 250ms for MS/MS acquired in the linear ion trap, with a CID energy settingof 35% and a dynamic exclusion of 60 s for previously fragmentedprecursor ions. Multistage activation (MSA) for neutral losses of 98.0,49.0, and 32.33 Da was enabled to enhance fragmentation ofphosphorylated peptides.

Non-phosphopeptide enriched data were acquired on a Waters Synapt HDMSoperating in positive-ion mode with an electrospray voltage of 3.0 kV.Each sample was analyzed three times in a data-independent (MS^(ε)) modeof acquisition with 0.9 sec cycle times alternating between lowcollision energy (6 V) and high collision energy ramp (15 to 40 V). Oneadditional data-dependent (DDA) analysis using a 0.9 sec MS scanfollowed by MS/MS acquisition on the top 3 ions with charge greater than1 was acquired to increase the number of qualitative identifications.MS/MS scans for each ion used an isolation window of approximately 3 Da,a maximum of 4 seconds per precursor, and dynamic exclusion for 120seconds within 1.2 Da.

Database Searching and Label-Free Quantitation.

Label-free quantitation and integration of qualitative peptideidentifications was performed using Rosetta Elucidator (v 3.3, RosettaInpharmatics, Seattle, Wash.). All raw LC-MS/MS data within either thephosphopeptide enriched or non-enriched experiments were imported andsubjected to chromatographic retention time alignment using thePeakTeller® algorithm with a minimum peak time width set to 6 s,alignment search distance set to 4 min and the refine alignment optionenabled. Quantitation of all measurable signals in the precursor MSspectra (excluding LC-MS analysis intended only for additionalqualitative identifications), was performed by Elucidator by calculatingeither peak volume (area under curve) for Synapt HDMS data files or peakheight for LTQ-Orbitrap data files.

Qualitative peptide identifications from all phosphopeptide enrichedsamples and DDA analysis of non-phosphopeptide enriched samples, weremade by generating DTA files for all precursor ions, which hadassociated MS/MS spectra. DTA files were submitted to Mascot (MatrixScience, Boston, Mass.) and searched against a Homo sapien proteindatabase downloaded from SwissProt concatenated with thesequence-reversed version of each entry. MS^(E) data were independentlyprocessed within ProteinLynx Global Server 2.4 (Waters Corp) andsearchable files were then submitted to the IdentityE search engine(Waters Corp). Search tolerances of 10 ppm precursor and 0.8 Da productions were initially applied to LTQ-Orbitrap data and then manuallyrefined to 4 ppm around the apex of the ppm mass error distribution fromthe most confident forward entries. Tolerances of 20 ppm precursor and0.04 Da product ions were applied for Synapt HDMS data files withlock-mass correction on m/z 785.8426 (doubly-chargedGlu-1-Fibrinopeptide ion) enabled. All data were searched using trypsinspecificity with up to two missed cleavages with a static modificationof Carbamidomethylation (+57.0214 Da on C) and dynamic modifications ofoxidation (+15.9949 Da on M). Dynamic search modifications ofphosphorylation (+79.9663 Da on STY) and of deamidation (+1.008 Da onNQ) were employed for phosphopeptide enriched sample andnon-phosphopeptide enriched samples, respectively. False-discovery ratewere determined by adjusting the Mascot peptide ion score threshold toallow a 1% occurrence of peptide spectral matches from reverse proteinentries for phosphopeptide enriched experiments, or by usingPeptideProphet algorithm thresholds corresponded to a 2% peptide falsediscovery rate for non-phosphopeptide enriched experiments.

Database search results and spectra have been uploaded in the form ofScaffold 3 files (.sf3, Proteome Software, Inc) to the Tranche database(https://proteomecommons.org/tranche/) under the group “RBC GhostMembrane Phosphoproteome” with the following links (if a password isrequested, it is rbcphos).

Glycophorin a Phosphorylation and Immunoprecipitation.

Packed RBCs ³²P-labeled as previously described,⁶⁸ were sham-treated, orincubated with serine/threonine phosphatase inhibitor (SPI) cocktail(Sigma) for 30 min, SPI cocktail followed by 1 min treatment with 20 nMepinephrine, or pre-incubated with 10 μM U0126 for 1 h followed by SPIcocktail, then treated with 20 nM epinephrine for 1 min. Cells were thenwashed 4 times. Glycophorin A immunoprecipitation using anti-glycophorinA monoclonal antibody (mAb) (Abcam, Cambridge, Mass.) and the negativecontrol immunoglobulin P3, and total and phospho-glycophorin A detectionwere performed as previously described in detail.⁶⁶ To confirm that theimmunoprecipitates were specific for glycophorin A, anti-glycophorin AmAb and the negative control P3 were used to immunoprecipitateglycophorin A from non-radiolabeled treated SS RBCs. Blots wereimmunostained with anti-glycophorin A mAb.

Statistical Analysis.

Data were compared using parametric analyses (GraphPad Prism 5 Software,San Diego, Calif.), including repeated and non-repeated measures ofanalysis of variance (ANOVA). One-way and two-way ANOVA analyses werefollowed by Bonferroni corrections for multiple comparisons (multiplyingthe p value by the number of comparisons). A p value <0.05 wasconsidered significant.

Label-Free Quantitative Phosphoproteomic Profiling of RBC Membranes.

Quantitation of global (non-targeted) phosphorylation events directlyfrom human RBCs in disease-affected patients has been very limited inthe literature. The most common analytical strategies have employedcoupling two-dimensional gel electrophoresis of solubilized RBC proteinswith either global ³²P labeling or anti-phosphotyrosine detectionantibodies, followed by LC-MS/MS identification of phosphoproteins fromdifferentially expressed protein spots. In addition to the limitednumber of unique treatment groups, which could be directly comparedwithin a single study, these previous approaches do not affordresidue-specific quantitation of phosphorylation events as initialdetection in changes in phosphorylation status measured at the proteinlevel. This is particularly problematic for proteins containing multiplesites of phosphorylation, as each could be independently modulated bydifferent kinases or phosphatases as a function of various stimuli. Inaddition, different phosphorylation sites could have different effect onprotein function. Although strategies such as iTRAQ, which are commonlyused for phosphoproteomic quantitation from non-cell culture basedsystems, address some of these limitations, the reagents are stilllimited to a maximum of eight unique treatment groups (or uniquesamples), and add significant cost when performing the labeling at thequantities of protein required for phosphoproteomic analysis.

Across all our samples tested, 375 unique phosphopeptides (527 totalphosphorylated residues) corresponding to 155 phosphoproteins wereidentified at a peptide spectral match false discovery rate of 1.0%. Aslocalization of specific phosphorylated residues is critical fordefining kinase specific events, all phosphopeptides were subjected toModLoc, a probability-based localization tool implemented within RosettaElucidator based on the AScore algorithm (FIG. 12A). (Beausoleil et al.,A probability-based approach for high-throughput protein phosphorylationanalysis and site localization. Nature biotechnology. October 2006;24(10):1285-1292). Approximately 74% (348) of phosphorylated residueshad ModLoc scores above 15 (>90% probability of correct localization)and 66% (310) had ModLoc scores above 20 (>99% probability of correctlocalization). To assess the quantitative robustness of the label-freeapproach, the average technical coefficient of variation (% CV) ofretention-time aligned phosphorylated peptide intensities within atreatment group were calculated. The average % CV across all 375phosphopeptides was 19.8%, with 80% of the signals having a % CVs lessthan 27.1% (FIG. 128). The intensity of the phosphorylated peptideV173-[pY187]-R191 within the activated site of ERK1/2 was used to assessinter-treatment group variation, including variation from TiO₂phosphopeptide enrichment, as activated ERK2 was spiked in equal amountsto four of the eight samples. The average % CV of this phosphopeptidewithin any treatment group was 7.0% and across all ERK2 spiked sampleswas 18.1% (FIGS. 13A&B).

Consistent with a majority of TiO₂-enrichment based global mammalianphosphoproteomic studies, 78% (415) of the identified phosphorylatedresidues were localized to serines, 16% (85) to threonines, and 5% (27)to tyrosines, with an average of 1.4 phosphorylated residues per peptide(FIG. 14A). Gene ontology classification of the biological function ofthe 155 identified phosphoproteins indicated nearly a third of thephosphoproteins were involved in binding as their primary biologicalfunction. Sub-classification of the binding category revealed over 80%of those phosphoproteins were involved in either protein binding (51%)or nucleotide binding (30%) (FIG. 14b ). Phosphoproteins involved in ionbinding consisted 12% of the total phosphoproteins. These data wereexpected since our RBC samples were membrane fractions. Consistent withother RBC membrane phosphorylation studies, the phosphoproteins of SSRBC membrane ghosts with the highest number of uniquely phosphorylatedpeptides (>10), were ankyrin-1 of the ankyrin complex (n=33), spectrin βchain of the cytoskeleton network (n=15), and proteins of the junctionalcomplex involved in binding integral membrane proteins to cytoskeletalproteins, including α- and β-adducins (n=22 and n=18, respectively),dematin (n=18) and protein 4.1 (n=17) (Table 2). However, with theexception of spectrin β chain, addusins α and β, and dematin, inhibitionof MEK1/2 and its downstream singling in SS RBCs with the MEK inhibitorU0126, and co-incubation of U0126-treated SS RBC membrane ghosts withactive recombinant ERK2, failed to induce significant changes (decreaseand increase, respectively) in the abundance of ankyrin-1 and protein4.1 phosphopeptides (Table 2).

TABLE 2 Unique phosphorylated SS RBC membrane proteomes. Unique UniqueGene Phosphorylated Phosphorylated Protein Description Name PeptidesResidues Ankyrin-1 ANK1 33 26 Glyophorin A GYPA 23 8 Alpha-adducin ADD122 22 Beta-adducin ADD2 18 Protein 4.1 EPB41 17 10 Dematin EPB49 16Spectrin beta chain SPTB1 15 15 Band 3 anion transport SLC4A1 14 10protein Uncharacterized protein YA047 7 LOC388588 GTPase-activatingprotein GAPVD1 5 and VPS9 domain- contaning protein 1 Lipin-2 LPIN2 5Serine/threonin-protein WNK1 5 kinase WNK1 Spectrin alpha chain SPTA1 4Erythroid membrane- ERMAP 4 associated protein Heat shock protein HSP90A4 HSP90-alpha Phosphatidylinositol PI4K2A 4 4-kinase TSC22 domain familyTSC22D4 4 protein 4Unique phosphopeptides and their unique phosphorylated residues in SSRBC membrane fractions are presented.

Previous studies have shown that protein 4.1 is extensivelyphosphorylated in sickle red cells. George, et al. Alteredphosphorylation of cytoskeleton proteins in sickle red blood cells: therole of protein kinase C, Rac GTPases, and reactive oxygen species.Blood Cells Mol Dis. Jun. 15 2010; 45(1):41-45. Protein 4.1phosphorylation, induced by cAMP-dependent kinase at Ser-331, results inmultiple changes in RBC membrane, including significant reduction bothin the ability of protein 4.1 to promote spectrin binding to F-actin andin spectrin-protein 4.1 binding. Ling et al., Modulation of red cellband 4.1 function by cAMP-dependent kinase and protein kinase Cphosphorylation. J Biol Chem. Feb. 15 1988; 263(5):2209-2216. Thesechanges weaken the binding sites for glycophorin C, X K and Duffy of the30 kDa domain and the stability of the ternary junction complex, withpossible effects on membrane mechanical stability and reduction in shearresistance to the membrane. However, while ERK1/2 signaling in SS RBCsis cAMP- and PKA-dependent, increased phosphorylation of protein 4.1 andankyrin-1 in SS RBCs seems to not involve ERK1/2 signaling. Our dataalso indicate that in addition to these commonly reportedphosphoproteins, several other phosphoproteins with >5 uniquephosphorylated peptides were also observed (Table 2). Thesephosphoproteins also affect RBC shape, flexibility, anion transport andprotein trafficking, and adhesion, all of which contribute to thepathophysiology of SCD.

ERK1/2 Induces Atypical Phosphorylation of SS RBC Membrane Proteins.

To assess global quantitative differences between all treatment groups,data were subjected to two-dimensional agglomerative clustering ofZ-score transformed (i.e. magnitude of significance of change)individual phosphopeptide intensities. This analysis revealed the mostsignificant differentiation (most negative Pearson correlation) acrossall treatment groups, was the sickle versus healthy red cell phenotype,with 201 phosphopeptides being significantly up-regulated in SS vs AARBCs at a p-value <0.05 and fold-increase of >1.75 (chosen based on analpha value corresponding to a 95% confidence interval in a statisticalpowering calculation). The weight of variation from the sickle state ofthe RBC (−0.664) was more significant than the addition of exogenousactive ERK2 or the inhibition of MEKI/2 activity with the MEK1/2inhibitor U0126, suggesting that in addition to MEK1/2/ERK1/2phosphorylation cascades in the SS RBC, other cellular signaling pathwayactivities are also involved (FIG. 15A). Interestingly, clustering ofall phosphopeptides within only the SS RBC samples revealed thestrongest differentiating factor was in the presence or absence of U0126(Pearson correlation, −0.422), which supports the previous observationthat ERK1/2 is constitutively hyperactive in these sickle RBCs andinhibiting ERK1/2's upstream activator, MEK1/2, alters a number ofsignaling events (FIGS. 15A&B). Recovery of the U1026 treatment byaddition of exogenous active ERK2 resulted in the phosphorylationprofile becoming more similar to the sham-treated SS RBCs. Incomparison, clustering of all phosphopeptides within only the AA RBCsamples revealed the strongest differentiating factor was the additionof exogenous active ERK2 (−0.489), which is consistent with the normalinactivity of ERK1/2 in AA RBCs and suggests that ERK1/2 signaling isindeed mediating downstream phosphorylation of a number of targets(FIGS. 15A&B).

Putative downstream targets specific to MEK1/2-dependent activation ofERK1/2 were initially identified comparing individual phosphopeptideintensities between SS RBCs and SS RBCs treated with U0126. The MEK1/2inhibitor U0126 was able to significantly down-regulate 36 unique RBCmembrane phosphopeptides (from 22 unique phosphoproteins) in SS RBCs(Table 3). We analyzed a number of these phosphoproteins referring firstto the model of red blood cell membrane functional organization proposedby Anong W A et al. who identified two major protein complexes bridgingthe RBC membrane to cytoskeleton network: the junctional complex formedby band 3, glycophorin C, Rh group, glucose transporter, dematin, p55,adducin, band 4.1 and 4.2 with associated glycolytic enzymes, and theankyrin complex formed by band 3, glycophorin A, Rh group, ankyrin, andprotein 4.2. Both complexes participate in anchoring the membrane to theactins, and α- and β-spectrins network, involving also other peripheralproteins as tropomyosin and tropomodulin. Here, we found thatMEK1/2-dependent ERK1/2 activation in SS RBCs affected membrane-boundproteomes of both the junctional and ankyrin complexes, includingdematin, α- and β-adducins, and glycophorin A. Glycophorin A was themost affected protein in SS RBCs by this pathway, which contained 11unique phosphorylated peptides with 8 unique phosphorylated residues (6phospho-serines and 2 phospho-threonines). The abundance of 6 of thephosphorylated residues, which was significantly downregulated withU0126 treatment of SS RBCs, was up-regulated in AA RBCs in the presenceof exogenous active ERK2, suggesting that increased phosphorylation ofglycophorin A by MEK1/2ERK1/2 signaling could potentially affect SS RBCmembrane properties. Glycophorin A, is the major sialoglycoprotein, andincreased SS RBC adhesion to vascular endothelial cells has beenpostulated to result from clustering of negatively chargedglycophorin-linked sialic acid moieties at the RBC surface. Enhanced SSRBC adhesion may also result from increased phosphorylation ofglycophorin A by MEK/1/2/ERK1/2 signaling. In addition, modulation inglycophorin A phosphorylation may also affect glycophorin A interactionswith band 3, which could result in decreased in both anion transport byband 3 and band 3 trafficking.

Our data also indicated that adducin-β contained three uniquephosphorylated peptides, with phosphorylation of residues within theERK1/2 consensus motif, suggesting that the cytoskeletal proteinadducin-β is a substrate for ERK1/2 in RBCs (Table 3). A significantdecrease in phosphorylation of these peptides was observed inU0126-treated SS RBCs, while a significant increase in phosphorylationwas observed in both U0126-treated SS RBCs and in AA RBCs whenrecombinant active ERK2 was added to the membrane ghosts. However, thephosphorylated serine on either adducin-α or dematin, was not within theERK1/2 consensus motif. Previous studies have shown that rapidphosphorylation of α- and β-adducins by PKC at Ser-726 and Ser-713,respectively, leads to decreased F-actin capping and dissociation ofspectrin from actin, implicating adducin phosphorylation in cytoskeletalremodeling. Alternatively, dematin is a substrate for PKC and PKA, andPKA-induced dematin phosphorylation completely abolishes its actinbundling capability. Studies in vitro and in vivo in mice geneticallylacking dematin have also shown its important role in maintaining redcell homeostasis and membrane mechanical properties.

TABLE 3 Changes in Phosphorylation of Peptides in SS and AA RBCs FoldChange: SS + Fold U0126 vs Change: SS + Fold SS vs U0126 + Change: SS +ERK AA vs U0126 (Relative AA + ERK Protein Modified (Relative p- to SS +p- (Relative p- Description Peptide to SS) value U0126) value to AA)value Leucine- MAGPGST*GGQIGAAAL −6.48 4.50E− 6.70 4.54E− zipper-likeAGGAR 02 06 transcrip- (SEQ ID NO: 8) tional regulator 1 AdenylylSGPKPFSAPKPQTS*PSP −4.79 2.00E− 5.33 2.00E− cyclase- K (5E0 ID NO. 9) 0304 associated protein 1 Dematin QPLTSPGSVS*PSR −4.40 1.00E−(SEQ ID NO: 10) 03 Alpha- QKGS*EENLDEAR −4.12 2.80E− 2.42 1.25E− adducin(SEQ ID NO: 11) 45 11 Protein VS*S*GIGAAAEVLVNLY −4.06 8.44E− MICAL-2*MNDHRPKAQAT*SPDL 05 ESMRK (SEQ ID NO: 12) facilitatedQGGAS*QSDKTPEELFHP −3.37 7.99E− glucose LGADSQV 08 transporter(SEQ ID NO: 13) member 1 U3 small VVHS*FDYAAS*ILSLALA −2.93 7.39E− 2.196.08E− 2.06 9.39E− nucleolar HEDETIVVGMTNGILS*V 11 05 14 RNA- KHRassociated (SEQ ID NO: 14) protein 15 Leucine- T*HPETIIALRGMNVTLTC −2.921.27E− 1.84 2.65E− rich re- TAVSSSDSPMST*VWR 23 05 peats and(SEQ ID NO: 15) immunoglo- bulin-like domains protein 2 Transmem-SPPGS*AAGES*AAGGG −2.85 3.91E− brane GGGGGPGVSEELTAAAA 05 proteinAAAADEGPAR 151B (SEQ ID NO: 16) Eukaryotic SQS*SDTEQQSPTSGGG −2.484.00E− 1.95 2.00E− translation K (SEQ ID NO: 17) 03 02 initiationfactor 4B Spectrin beta QIAERPAEETGPQEEEGE −2.42 5.77E− chain,TAGEAPVS*HHAATER 04 erythrocyte (SEQ ID NO: 18) Glycophorin-AKSPSDVKPLPS*PDT*DV −2.26 5.00E− 2.12 8.04E− PLS*SVEIENPETSDQ 03 04(SEQ ID NO: 19) Glycophorin-A SPSDVKPLPSPDTDVPLS* −2.18 3.00E− 2.747.97E− S*VEIENPETSDQ V 03 05 (SEQ ID NO: 20) 60S acidicKEES*EES*DDDMGFGLF −2.11 3.61E− ribosomal D (SEQ ID NO: 21) 08protein P2 Glycophorin-A SPSDVKPLPSPDT*DVPLS −2.00 2.59E− SVEIENPETSDQ21 (SEQ ID NO: 22) Glycophorin-A S*PS*DVKPLPSPDTDVPL −1.99 1.60E− 1.873.10E− SSVEIENPETS*DQ 02 02 (SEQ ID NO: 23) Glycophorin-AS*PS*DVKPLPSPDTDVPL −1.98 8.00E− SSVEIENPETSDQ 03 (SEQ ID NO: 24)Glycophorin-A SPSDVKRLPS*PDT*DVPL −1.96 2.13E− SSVEIENPETSDQ 07(SEQ ID NO: 25) Protein Wnt- HERWNCMITAAATTAP −1.95 1.90E− 16MGASPLFGYELS*SGTK 02 (SEQ ID NO: 26) UV excision EDKS*PSEESAPTTSPESV−1.95 1.96E− 2.19 4.13E− repair SGSVPSSGSSGR 14 32 protein(SEQ ID NO: 27) RAD23 homolog A Glycophorin-A SPSDVKPLPSPDTDVPLSS −1.931.82E− VEIENPET*SDQ 13 (SEQ ID NO: 28) Metabotropic LSHKPSDRPNGEAKT*EL−1.91 2.95E− 2.29 7.98E− glutamate CENVDPNS*PAAK 09 15 receptor 7(SEQ ID NO: 29) Beta-adducin ETAPEEPGS*PAKS*APA −1.91 3.87E− 2.36 4.21E−S*PVQSPAK 09 06 (SEQ ID NO: 30) Glycophorin-A KS*PSDVKPLPSPDTDVP −1.882.10E− 1.97 5.00E− LSSVEIENPETS*DQ 02 03 (SEQ ID NO: 31) Beta-adducinTESVTSGPMSPEGSPSKS −1.88 3.00E− 1.85 3.00E− *PSK  02 03 (SEQ ID NO: 32)Glycophorin-A SPSDVKPLPSPDTDVPLSS −1.87 3.30E− *VEIENPETSDQ 07(SEQ ID NO: 33) Lipin-2 S*GGDETPSQSSDISHVL −1.86 3.37E− ETETIFTPSSVK 04(SEQ ID NO: 34) Proteasome ESLKEEDES*DDDNM −1.83 5.42E− $uhunit(SEQ ID NO: 35) 06 alpha type-3 Beta-adducin ETAPEEPGSPAKS*APAS −1.821.80E− 1.80 3.00E− 1.75 2.00E− *PVQSPAK 02 02 02 (SEQ ID NO: 36)E3 obiquitin- T*SPADHGGSVGSESGG −1.80 1.02E− protein ligaseSAVDSVAGEHSVSGR 07 UBR4 (SEQ ID NO: 37) Spectrin beta LS*S*SWES*LQPEPSHP−1.80 6.00E− 4.41 4.02E− chain, Y (SEQ ID NO: 38) 03 21 erythtocyteUncharacterized DGVS*LGAVSST*EEASR −1.80 4.90E− protein (SEQ ID NO: 39)02 LOC388588 Glycophorin-A SPSDVKPLPSPDTDVPLSS −1.77 4.00E− VEIENPETS*DQ03 (SEQ ID NO: 40) Spectrin beta LS*SS*WESLQPEPSHPY −1.76 5.56E− 2.029.30E− chain, (SEQ ID NO: 41) 05 11 erythrocyte UncharacterizedDGVS*LGAVS*STEEASR −1.76 9.99E− 2.19 2.40E− protein (SEQ ID NO: 42) 0509 LOC388588 Glycophorin-A SPSDVKPLPSPDT*DVPLS −1.75 4.80E−*SVEIENPETSDQ 02 (SEQ ID NO: 43) *Indicates site of phosphorylation

MEK1/2/ERK1/2 signaling in SS RBCs induced changes within theactins/spectrins network as well, by affecting phosphorylation ofβ-spectrins (Table 3). Erythrocyte spectrin, the major component of themembrane skeleton, undergoes a number of naturally occurring orpathologically induced posttranslational phosphorylation via acAMP-dependent protein kinase. ³²P-labeling studies indicate that onlythe α-subunit of spectrin is phosphorylated in intact erythrocyte, andphosphorylation occurs in a sequential manner where each specific siteis completely phosphorylated before the next site is modified with thefirst phosphorylation event occurring on Ser-2114, followed by Ser-2125,Ser-2123, Ser-2128, Ser-2117, and Ser-2110. However, in situ studies byManno et al. using intact erythrocyte membranes demonstrated that anincrease in β-spectrin phosphorylation by casein kinase I causes adecrease in erythrocyte membrane mechanical stability. In addition,certain leukemia patients with elliptocytosis and poikilocytosisdisplayed an elevated amount of spectrin dimers coinciding withincreased β-spectrin phosphorylation. Our findings are in accordancewith these previous studies, and all together strongly suggest thatincreased phosphorylation of β-spectrin destabilizes tetramer formationand has important in vivo physiological functions. Membrane skeletonalso appears to regulate lateral and rotational mobility of band 3 andglycophorin A in the plane of the membrane.

Furthermore and interestingly, label-free proteomic analysis revealedthat the peptide metabotropic glutamate receptor 7 (mGlu7) underwentserine phosphorylation at the ERK consensus motif (Table 3). Indeed,studies have also demonstrated that mGluR7 activation occurs via anERK-dependent mechanism, which increased cofilin activity and F-actindepolymerization. mGLu7 acts as an autoreceptor mediating the feedbackinhibition of glutamate release, and prolonged activation of thisreceptor potentiates glutamate release. Increased phosphorylation ofmGlu7 in SS RBCs, could explain the rate of active glutamate transportin these cells, which increases 15-fold over that in normal RBCs.Significant Changes were also observed in the status of leucine-richrepeats and immunoglobulin-like domains protein 2, leucine-zipper-liketranscriptional regulator 1, and glucose transporter 1, but only inmembrane ghosts prepared from SS RBCs treated with U0126 or afteraddition of exogenous active ERK2 to these membrane ghosts (Table 3).Changes in the status of these proteins by MEK1/2/ERK1/2 signaling maypotentially disturb degradation of misfolded glycoproteins and receptorubiquitination, and affect protein transcription. Similarly and notsurprisingly, ERK1/2 signaling was also found to increase phosphorylatedadenylyl cyclase-associated protein 1 (CAP1) only in SS RBCs. CAP1 isknown to regulate adenylate cyclase activation to increase cAMP levelsunder specific environmental conditions. Indeed, basal cAMP levels aremuch higher in sickle than in healthy RBCs, and cAMP and PKA can act asupstream effectors of MEK1/2/ERK1/2 in SS RBCs. CAPs are also involvedin actin binding, SH3 binding, and cell morphology maintenance as well.The failure of recombinant active ERK2 to significantly upregulate theabundance of the phosphorylated peptides, leucine-rich repeats andimmunoglobulin-like domains protein 2, leucine-zipper-liketranscriptional regulator 1 and CAP1, in healthy RBCs suggests anegative regulatory mechanism might exist in these cells to preventactivation of ERK1/2-dependent phosphorylation of these membraneproteins. PKA for instance, has been shown to exert a negative feedbackloop through activation of phosphodiesterases, resulting in CAMPhydrolysis switching off downstream signaling.

ERK1/2 is Involved in Phosphorylation of Glycophorin A.

The pharmacological stress hormone epinephrine can modulate ERK1/2activation in SS RBCs. Because our proteomics data showed thatERK1/2-induced changes in the phosphorylation state of glycophorin Aaffected numerous peptides, we determined the contribution ofepinephrine-induced increased activation of ERK1/2 signaling inglycophorin A phosphorylation. PhosphorImager analysis ofimmunoprecipitated ³²P-radiolabeled glycophorin A and negative controlimmune complexes showed that glycophorin A of non-stimulated SS RBCs(FIG. 16, lane 1) is modestly phosphorylated at baseline. Treatment ofSS RBCs with serine phosphatase inhibitors (SPI) (FIG. 16, lane 2)slightly increased glycophorin A phosphorylation by 1.9±0.1-fold(p<0.05, n=3), suggesting that increased glycophorin A phosphorylationis a result of serine phosphorylation, as tyrosine phosphataseinhibitors were not present. Epinephrine in the presence of SPI had astronger effect on glycophorin A phosphorylation (2.93±0.35-foldincrease over sham-treated SS RBCs; p<0.001) (FIG. 16; lane 3). However,treatment of SS RBCs with the MEK/12 inhibitor U0126 significantlydecreased the combined effect of epinephrine and SPI on glycophorin Aphosphorylation (FIG. 16; lane 4) compared to cells treated withepinephrine and SPI (p<0.001) (FIG. 16; lane 3). Immunoblots of³²P-radiolabeled glycophorin A immunoprecipitates from stimulated andnon-stimulated SS RBCs indicated that a similar amount of glycophorin Awas immunoprecipitated from these cells. Our data strongly confirm thatincreased glycophorin A phosphorylation is dependent on MEK1/2-dependentERK1/2 signaling pathway in SS5 RBCs. It has been suggested thatglycophorin contains receptors or other surface recognition sites of theerythrocyte. Although the conformation of glycophorin in the lipidbilayer is not known, it has been suggested that the glycoproteins existas aggregates in the membrane in order to facilitate receptor function.However, we do not know whether increased phosphorylation of glycophorinA affects the state of aggregation of this glycoprotein. Recently,Shapiro and Marchesi have demonstrated that the site of phosphorylationof glycophorin is located on the cytoplasmic COOH terminal end.⁶⁵ Itremains to be determined if phosphorylation plays a role in theformation of aggregates of the protein.

Indeed, ERK activation in sickle RBCs not only up-regulated sickle redcell adhesion to TNF-α activated endothelial cells in vitro, butaffected proteins involved in nitric oxide transport, oxidative stress,proteins of the water channel, maintenance of the integrity of theplasma membrane and to anchor specific ion channels, ion exchangers andion transporters in the plasma membrane, membrane morphogenesis andcytoskeletal organization, regulation of integrin-mediated signaling,and membrane integrity, permeability and polarity as well (Table 3). TheMEK inhibitor U0126 down-regulates phosphorylation of ERK targets. Thesedata suggest that ERK is involved not only in abnormal SS RBC adhesion,but affects multiple other red cell functions related but not limited tooxidative stress, hemolysis and ion transport.

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The invention claimed is:
 1. A method of alleviating vaso-occlusion in apatient with a hemoglobinopathy comprising administering to the patienta MEK inhibitor; wherein the patient has experienced at least onevaso-occlusive event, and wherein the MEK inhibitor is selected fromU0126, PD98059, PD-334581, GDC-0973, CIP-137401, ARRY-162, ARRY-300,PD318088, PD0325901, CI-1040, BMS 777607, AZD8330, AZD6244, AS703026,RDEA119, and GSK1120212.
 2. The method of claim 1, wherein thehemoglobinopathy is selected from sickle cell disease, β-thalassemia,and hemoglobin H disease.
 3. The method of claim 2, wherein thehemoglobinopathy is sickle cell disease.
 4. The method of claim 1,wherein the patient is human.
 5. The method of claim 1, wherein theinhibitor is administered to the patient while the patient isexperiencing a vaso-occlusive event.
 6. The method of claim 1, whereinthe inhibitor is administered to the patient while the patient is notexperiencing a vaso-occlusive event.